Meningococcal antigens

ABSTRACT

The invention provides proteins from  Neisseria meningitidis  (strains A &amp; B), including amino acid sequences, the corresponding nucleotide sequences, expression data, and serological data. The proteins are useful antigens for vaccines, immunogenic compositions, and/or diagnostics.

This application is a continuation-in-part of international patent application PCT/IB99/00103, filed Jan. 14, 1999, from which priority is claimed under 35 U.S.C. § 119.

This invention relates to antigens from the bacterium Neisseria meningitidis.

BACKGROUND

Neisseria meningitidis is a non-motile, gram negative diplococcus human pathogen. It colonises the pharynx, causing meningitis and, occasionally, septicaemia in the absence of meningitis. It is closely related to N. gonorrhoeae, although one feature that clearly differentiates meningococcus from gonococcus is the presence of a polysaccharide capsule that is present in all pathogenic meningococci.

N. meningitidis causes both endemic and epidemic disease. In the United States the attack rate is 0.6-1 per 100,000 persons per year, and it can be much greater during outbreaks (see Lieberman el al. (1996) Safety and Immunogenicity of a Serogroups A/C Neisseria meningitidis Oligosaccharide-Protein Conjugate Vaccine in Young Children. JAMA 275(19):1499-1503; Schuchat et al (1997) Bacterial Meningitis in the United States in 1995. N Engl J Med 337(14):970-976). In developing countries, endemic disease rates are much higher and during epidemics incidence rates can reach 500 cases per 100,000 persons per year. Mortality is extremely high, at 10-20% in the United States, and much higher in developing countries. Following the introduction of the conjugate vaccine against Haemophilus influenzae, N. meningitidis is the major cause of bacterial meningitis at all ages in the United States (Schuchat et al (1997) supra).

Based on the organism's capsular polysaccharide, 12 serogroups of N. meningitidis have been identified. Group A is the pathogen most often implicated in epidemic disease in sub-Saharan Africa. Serogroups B and C are responsible for the vast majority of cases in the United States and in most developed countries. Serogroups W135 and Y are responsible for the rest of the cases in the United States and developed countries. The meningococcal vaccine currently in use is a tetravalent polysaccharide vaccine composed of serogroups A, C, Y and W135. Although efficacious in adolescents and adults, it induces a poor immune response and short duration of protection, and cannot be used in infants [eg. Morbidity and Mortality weekly report, Vol. 46, No. RR-5 (1997)]. This is because polysaccharides are T-cell independent antigens that induce a weak immune response that cannot be boosted by repeated immunization. Following the success of the vaccination against H. influenzae, conjugate vaccines against serogroups A and C have been developed and are at the final stage of clinical testing (Zollinger W D “New and Improved Vaccines Against Meningococcal Disease” in: New Generation Vaccines, supra, pp. 469-488; Lieberman et al (1996) supra; Costantino et al (1992) Development and phase I clinical testing of a conjugate vaccine against meningococcus A and C. Vaccine 10:691-698).

Meningococcus B remains a problem, however. This serotype currently is responsible for approximately 50% of total meningitis in the United States, Europe, and South America. The polysaccharide approach cannot be used because the menB capsular polysaccharide is a polymer of α(2-8)-linked N-acetyl neuraminic acid that is also present in mammalian tissue. This results in tolerance to the antigen; indeed, if an immune response were elicited, it would be anti-self, and therefore undesirable. In order to avoid induction of autoimmunity and to induce a protective immune response, the capsular polysaccharide has, for instance, been chemically modified substituting the N-acetyl groups with N-propionyl groups, leaving the specific antigenicity unaltered (Romero & Outschoorn (1994) Current status of Meningococcal group B vaccine candidates: capsular or non-capsular? Clin Microbiol Rev 7(4):559-575).

Alternative approaches to menB vaccines have used complex mixtures of outer membrane proteins (OMPs), containing either the OMPs alone, or OMPs enriched in porins, or deleted of the class 4 OMPs that are believed to induce antibodies that block bactericidal activity. This approach produces vaccines that are not well characterized. They are able to protect against the homologous strain, but are not effective at large where there are many antigenic variants of the outer membrane proteins. To overcome the antigenic variability, multivalent vaccines containing up to nine different porins have been constructed (eg. Poolman J T (1992) Development of a meningococcal vaccine. Infect. Agents Dis. 4:13-28). Additional proteins to be used in outer membrane vaccines have been the opa and opc proteins, but none of these approaches have been able to overcome the antigenic variability (eg. Ala'Aldeen & Borriello (1996) The meningococcal transferrin-binding proteins 1 and 2 are both surface exposed and generate bactericidal antibodies capable of killing homologous and heterologous strains. Vaccine 14(1):49-53).

A certain amount of sequence data is available for meningococcal and gonococcal genes and proteins (eg. EP-A-0467714, WO96/29412), but this is by no means complete. The provision of further sequences could provide an opportunity to identify secreted or surface-exposed proteins that are presumed targets for the immune system and which are not antigenically variable. For instance, some of the identified proteins could be components of efficacious vaccines against meningococcus B, some could be components of vaccines against all meningococcal serotypes, and others could be components of vaccines against all pathogenic Neisseriae.

The Invention

The invention provides proteins comprising the N. meningitidis amino acid sequences disclosed in the examples.

It also provides proteins comprising sequences homologous (ie. having sequence identity) to the N. meningitidis amino acid sequences disclosed in the examples. Depending on the particular sequence, the degree of sequence identity is preferably greater than 50% (eg. 60%, 70%, 80%, 90%, 95%, 99% or more). These homologous proteins include mutants and allelic variants of the sequences disclosed in the examples. Typically, 50% identity or more between two proteins is considered to be an indication of functional equivalence. Identity between the proteins is preferably determined by the Smith-Waterman homology search algorithm as implemented in the MPSRCH program (Oxford Molecular), using an affine gap search with parameters gap open penalty=12 and gap extension penalty=1.

The invention further provides proteins comprising fragments of the N. meningitidis amino acid sequences disclosed in the examples. The fragments should comprise at least n consecutive amino acids from the sequences and, depending on the particular sequence, n is 7 or more (eg. 8, 10, 12, 14, 16, 18, 20 or more). Preferably the fragments comprise an epitope from the sequence.

The proteins of the invention can, of course, be prepared by various means (eg. recombinant expression, purification from cell culture, chemical synthesis etc.) and in various forms (eg. native, fusions etc.). They are preferably prepared in substantially pure form (ie. substantially free from other N. meningitidis or host cell proteins).

According to a further aspect, the invention provides antibodies which bind to these proteins. These may be polyclonal or monoclonal and may be produced by any suitable means.

According to a further aspect, the invention provides nucleic acid comprising the N. meningitidis nucleotide sequences disclosed in the examples. In addition, the invention provides nucleic acid comprising sequences homologous (ie. having sequence identity) to the N. meningitidis nucleotide sequences disclosed in the examples.

Furthermore, the invention provides nucleic acid which can hybridise to the N. meningitidis nucleic acid disclosed in the examples, preferably under “high stringency” conditions (eg. 65° C. in a 0.1×SSC, 0.5% SDS solution).

Nucleic acid comprising fragments of these sequences are also provided. These should comprise at least n consecutive nucleotides from the N. meningitidis sequences and, depending on the particular sequence, n is 10 or more (eg 12, 14, 15, 18, 20, 25, 30, 35, 40 or more).

According to a further aspect, the invention provides nucleic acid encoding the proteins and protein fragments of the invention.

It should also be appreciated that the invention provides nucleic acid comprising sequences complementary to those described above (eg. for antisense or probing purposes).

Nucleic acid according to the invention can, of course, be prepared in many ways (eg. by chemical synthesis, from genomic or cDNA libraries, from the organism itself etc.) and can take various forms (eg. single stranded, double stranded, vectors, probes etc.).

In addition, the term “nucleic acid” includes DNA and RNA, and also their analogues, such as those containing modified backbones, and also peptide nucleic acids (PNA) etc.

According to a further aspect, the invention provides vectors comprising nucleotide sequences of the invention (eg. expression vectors) and host cells transformed with such vectors.

According to a further aspect, the invention provides compositions comprising protein, antibody, and/or nucleic acid according to the invention. These compositions may be suitable as vaccines, for instance, or as diagnostic reagents, or as immunogenic compositions.

The invention also provides nucleic acid, protein, or antibody according to the invention for use as medicaments (eg. as vaccines) or as diagnostic reagents. It also provides the use of nucleic acid, protein, or antibody according to the invention in the manufacture of: (i) a medicament for treating or preventing infection due to Neisserial bacteria; (ii) a diagnostic reagent for detecting the presence of Neisserial bacteria or of antibodies raised against Neisserial bacteria; and/or (iii) a reagent which can raise antibodies against Neisserial bacteria Said Neisserial bacteria may be any species or strain (such as N. gonorrhoeae) but are preferably N. meningitidis, especially strain A, strain B or strain C.

The invention also provides a method of treating a patient, comprising administering to the patient a therapeutically effective amount of nucleic acid, protein, and/or antibody according to the invention.

According to further aspects, the invention provides various processes.

A process for producing proteins of the invention is provided, comprising the step of culturing a host cell according to the invention under conditions which induce protein expression.

A process for producing protein or nucleic acid of the invention is provided, wherein the protein or nucleic acid is synthesised in part or in whole using chemical means.

A process for detecting polynucleotides of the invention is provided, comprising the steps of: (a) contacting a nucleic probe according to the invention with a biological sample under hybridizing conditions to form duplexes; and (b) detecting said duplexes.

A process for detecting proteins of the invention is provided, comprising the steps of: (a) contacting an antibody according to the invention with a biological sample under conditions suitable for the formation of an antibody-antigen complexes; and (b) detecting said complexes.

Unlike the sequences disclosed in PCT/IB98/01665, the sequences disclosed in the present application are believed not to have any significant homologs in N. gonorrhoeae. Accordingly, the sequences of the present invention also find use in the preparation of reagents for distinguishing between N. meningitidis and N. gonorrhoeae.

A summary of standard techniques and procedures which may be employed in order to perform the invention (eg. to utilise the disclosed sequences for vaccination or diagnostic purposes) follows. This summary is not a limitation on the invention but, rather, gives examples that may be used, but are not required.

General

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature eg. Sambrook Molecular Cloning; A Laboratory Manual, Second Edition (1989); DNA Cloning, Volumes I and ii (D. N Glover ed. 1985); Oligonucleotide Synthesis (M. J. Gait ed, 1984); Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds. 1984); Transcription and Translation (B. D. Hames & S. J. Higgins eds. 1984); Animal Cell Culture (R. I. Freshney ed. 1986); Immobilized Cells and Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide to Molecular Cloning (1984); the Methods in Enzymology series (Academic Press, Inc.), especially volumes 154 & 155; Gene Transfer Vectors for Mammalian Cells (J. H. Miller and M. P. Calos eds. 1987, Cold Spring Harbor Laboratory); Mayer and Walker, eds. (1987), Immunochemical Methods in Cell and Molecular Biology (Academic Press, London); Scopes, (1987) Protein Purification: Principles and Practice, Second Edition (Springer-Verlag, N.Y.), and Handbook of Experimental Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell eds 1986).

Standard abbreviations for nucleotides and amino acids are used in this specification.

All publications, patents, and patent applications cited herein are incorporated in full by reference. In particular, the contents of UK patent applications 9800760.2, 9819015.0 and 9822143.5 are incorporated herein.

Definitions

A composition containing X is “substantially free of” Y when at least 85% by weight of the total X+Y in the composition is X. Preferably, X comprises at least about 90% by weight of the total of X+Y in the composition, more preferably at least about 95% or even 99% by weight.

The term “comprising” means “including” as well as “consisting” eg. a composition “comprising” X may consist exclusively of X or may include something additional to X, such as X+Y.

A “conserved” Neisseria amino acid fragment or protein is one that is present in a particular Neisserial protein in at least x % of Neisseria. The value of x may be 50% or more, e.g., 66%, 75%, 80%, 90%, 95% or even 100% (i.e. the amino acid is found in the protein in question in all Neisseria). In order to determine whether an animo acid is “conserved” in a particular Neisserial protein, it is necessary to compare that amino acid residue in the sequences of the protein in question from a plurality of different Neisseria (a reference population). The reference population may include a number of different Neisseria species or may include a single species. The reference population may include a number of different serogroups of a particular species or a single serogroup. A preferred reference population consists of the 5 most common Neisseria.

The term “heterologous” refers to two biological components that are not found together in nature. The components may be host cells, genes, or regulatory regions, such as promoters. Although the heterologous components are not found together in nature, they can function together, as when a promoter heterologous to a gene is operably linked to the gene. Another example is where a Neisserial sequence is heterologous to a mouse host cell. A further examples would be two epitopes from the same or different proteins which have been assembled in a single protein in an arrangement not found in nature.

An “origin of replication” is a polynucleotide sequence that initiates and regulates replication of polynucleotides, such as an expression vector. The origin of replication behaves as an autonomous unit of polynucleotide replication within a cell, capable of replication under its own control. An origin of replication may be needed for a vector to replicate in a particular host cell. With certain origins of replication, an expression vector can be reproduced at a high copy number in the presence of the appropriate proteins within the cell. Examples of origins are the autonomously replicating sequences, which are effective in yeast; and the viral T-antigen, effective in COS-7 cells.

A “mutant” sequence is defined as DNA, RNA or amino acid sequence differing from but having sequence identity with the native or disclosed sequence. Depending on the particular sequence, the degree of sequence identity between the native or disclosed sequence and the mutant sequence is preferably greater than 50% (eg. 60%, 70%, 80%, 90%, 95%, 99% or more, calculated using the Smith-Waterman algorithm as described above). As used herein, an “allelic variant” of a nucleic acid molecule, or region, for which nucleic acid sequence is provided herein is a nucleic acid molecule, or region, that occurs essentially at the same locus in the genome of another or second isolate, and that, due to natural variation caused by, for example, mutation or recombination, has a similar but not identical nucleic acid sequence. A coding region allelic variant typically encodes a protein having similar activity to that of the protein encoded by the gene to which it is being compared. An allelic variant can also comprise an alteration in the 5′ or 3′ untranslated regions of the gene, such as in regulatory control regions (eg. see U.S. Pat. No. 5,753,235).

Expression Systems

The Neisserial nucleotide sequences can be expressed in a variety of different expression systems; for example those used with mammalian cells, baculoviruses, plants, bacteria, and yeast.

i. Mammalian Systems

Mammalian expression systems are known in the art. A mammalian promoter is any DNA sequence capable of binding mammalian RNA polymerase and initiating the downstream (3′) transcription of a coding sequence (eg. structural gene) into mRNA. A promoter will have a transcription initiating region, which is usually placed proximal to the 5′ end of the coding sequence, and a TATA box, usually located 25-30 base pairs (bp) upstream of the transcription initiation site. The TATA box is thought to direct RNA polymerase II to begin RNA synthesis at the correct site. A mammalian promoter will also contain an upstream promoter element, usually located within 100 to 200 bp upstream of the TATA box. An upstream promoter element determines the rate at which transcription is initiated and can act in either orientation [Sambrook et al. (1989) “Expression of Cloned Genes in Mammalian Cells.” In Molecular Cloning: A Laboratory Manual, 2nd ed.]

Mammalian viral genes are often highly expressed and have a broad host range; therefore sequences encoding mammalian viral genes provide particularly useful promoter sequences. Examples include the SV40 early promoter, mouse mammary tumor virus LTR promoter, adenovirus major late promoter (Ad MLP), and herpes simplex virus promoter. In addition, sequences derived from non-viral genes, such as the murine metallotheionein gene, also provide useful promoter sequences. Expression may be either constitutive or regulated (inducible), depending on the promoter can be induced with glucocorticoid in hormone-responsive cells.

The presence of an enhancer element (enhancer), combined with the promoter elements described above, will usually increase expression levels. An enhancer is a regulatory DNA sequence that can stimulate transcription up to 1000-fold when linked to homologous or heterologous promoters, with synthesis beginning at the normal RNA start site. Enhancers are also active when they are placed upstream or downstream from the transcription initiation site, in either normal or flipped orientation, or at a distance of more than 1000 nucleotides from the promoter [Maniatis et al. (1987) Science 236:1237; Alberts et al. (1989) Molecular Biology of the Cell, 2nd ed.]. Enhancer elements derived from viruses may be particularly useful, because they usually have a broader host range. Examples include the SV40 early gene enhancer [Dijkema et al (1985) EMBO J. 4:761] and the enhancer/promoters derived from the long terminal repeat (LTR) of the Rous Sarcoma Virus [Gorman et al. (1982b) Proc. Natl. Acad. Sci. 79:6777] and from human cytomegalovirus [Boshart et al. (1985) Cell 41:521]. Additionally, some enhancers are regulatable and become active only in the presence of an inducer, such as a hormone or metal ion [Sassone-Corsi and Borelli (1986) Trends Genet. 2:215; Maniatis et al. (1987) Science 236:1237].

A DNA molecule may be expressed intracellularly in mammalian cells. A promoter sequence may be directly linked with the DNA molecule, in which case the first amino acid at the N-terminus of the recombinant protein will always be a methionine, which is encoded by the ATG start codon. If desired, the N-terminus may be cleaved from the protein by in vitro incubation with cyanogen bromide.

Alternatively, foreign proteins can also be secreted from the cell into the growth media by creating chimeric DNA molecules that encode a fusion protein comprised of a leader sequence fragment that provides for secretion of the foreign protein in mammalian cells. Preferably, there are processing sites encoded between the leader fragment and the foreign gene that can be cleaved either in vivo or in vitro. The leader sequence fragment usually encodes a signal peptide comprised of hydrophobic amino acids which direct the secretion of the protein from the cell. The adenovirus triparite leader is an example of a leader sequence that provides for secretion of a foreign protein in mammalian cells.

Usually, transcription termination and polyadenylation sequences recognized by mammalian cells are regulatory regions located 3′ to the translation stop codon and thus, together with the promoter elements, flank the coding sequence. The 3′ terminus of the mature mRNA is formed by site-specific post-transcriptional cleavage and polyadenylation [Birnstiel et al. (1985) Cell 41:349; Proudfoot and Whitelaw (1988) “Termination and 3′ end processing of eukaryotic RNA. In Transcription and splicing (ed. B. D. Hames and D. M. Glover); Proudfoot (1989) Trends Biochem. Sci. 14:105]. These sequences direct the transcription of an mRNA which can be translated into the polypeptide encoded by the DNA. Examples of transcription terminater/polyadenylation signals include those derived from SV40 [Sambrook et al (1989) “Expression of cloned genes in cultured mammalian cells.” In Molecular Cloning: A Laboratory Manual].

Usually, the above described components, comprising a promoter, polyadenylation signal, and transcription termination sequence are put together into expression constructs. Enhancers; introns with functional splice donor and acceptor sites, and leader sequences may also be included in an expression construct, if desired. Expression constructs are often maintained in a replicon, such as an extrachromosomal element (eg. plasmids) capable of stable maintenance in a host, such as mammalian cells or bacteria. Mammalian replication systems include those derived from animal viruses, which require trans-acting factors to replicate. For example, plasmids containing the replication systems of papovaviruses, such as SV40 [Gluzman (1981) Cell 23:175] or polyomavirus, replicate to extremely high copy number in the presence of the appropriate viral T antigen. Additional examples of mammalian replicons include those derived from bovine papillomavirus and Epstein-Barr virus. Additionally, the replicon may have two replicaton systems, thus allowing it to be maintained, for example, in mammalian cells for expression and in a prokaryotic host for cloning and amplification. Examples of such mammalian-bacteria shuttle vectors include pMT2 [Kaufman et al. (1989) Mol. Cell. Biol. 9:946] and pHEBO [Shimizu et al. (1986) Mol. Cell. Biol. 6:1074].

The transformation procedure used depends upon the host to be transformed. Methods for introduction of heterologous polynucleotides into mammalian cells are known in the art and include dextran-mediated transfection, calcium phosphate precipitation, polybrene mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide(s) in liposomes, and direct microinjection of the DNA into nuclei.

Mammalian cell lines available as hosts for expression are known in the art and include many immortalized cell lines available from the American Type Culture Collection (ATCC), including but not limited to, Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (eg. Hep G2), and a number of other cell lines.

ii. Baculovirus Systems

The polynucleotide encoding the protein can also be inserted into a suitable insect expression vector, and is operably linked to the control elements within that vector. Vector construction employs techniques which are known in the art. Generally, the components of the expression system include a transfer vector, usually a bacterial plasmid, which contains both a fragment of the baculovirus genome, and a convenient restriction site for insertion of the heterologous gene or genes to be expressed; a wild type baculovirus with a sequence homologous to the baculovirus-specific fragment in the transfer vector (this allows for the homologous recombination of the heterologous gene in to the baculovirus genome); and appropriate insect host cells and growth media.

After inserting the DNA sequence encoding the protein into the transfer vector, the vector and the wild type viral genome are transfected into an insect host cell where the vector and viral genome are allowed to recombine. The packaged recombinant-virus is expressed and recombinant plaques are identified and purified. Materials and methods for baculovirus/insect cell expression systems are commercially available in kit form from, inter alia, Invitrogen, San Diego Calif. (“MaxBac” kit). These techniques are generally known to those skilled in the art and fully described in Summers and Smith, Texas Agricultural Experiment Station Bulletin No. 1555 (1987) (hereinafter “Summers and Smith”).

Prior to inserting the DNA sequence encoding the protein into the baculovirus genome, the above described components, comprising a promoter, leader (if desired), coding sequence of interest, and transcription termination sequence, are usually assembled into an intermediate transplacement construct (transfer vector). This construct may contain a single gene and operably linked regulatory elements; multiple genes, each with its owned set of operably linked regulatory elements; or multiple genes, regulated by the same set of regulatory elements. Intermediate transplacement constructs are often maintained in a replicon, such as an extrachromosomal element (eg. plasmids) capable of stable maintenance in a host, such as a bacterium. The replicon will have a replication system, thus allowing it to be maintained in a suitable host for cloning and amplification.

Currently, the most commonly used transfer vector for introducing foreign genes into AcNPV is pAc373. Many other vectors, known to those of skill in the art, have also been designed. These include, for example, pVL985 (which alters the polyhedrin start codon from ATG to ATT, and which introduces a BamHI cloning site 32 basepairs downstream from the ATT; see Luckow and Summers, Virology (1989) 17:31.

The plasmid usually also contains the polyhedrin polyadenylation signal (Miller et al. (1988) Ann. Rev. Microbiol., 42:177) and a prokaryotic ampicillin-resistance (amp) gene and origin of replication for selection and propagation in E. coli.

Baculovirus transfer vectors usually contain a baculovirus promoter. A baculovirus promoter is any DNA sequence capable of binding a baculovirus RNA polymerase and initiating the downstream (5′ to 3) transcription of a coding sequence (eg. structural gene) into mRNA. A promoter will have a transcription initiation region which is usually placed proximal to the 5′ end of the coding sequence. This transcription initiation region usually includes an RNA polymerase binding site and a transcription initiation site. A baculovirus transfer vector may also have a second domain called an enhancer, which, if present, is usually distal to the structural gene. Expression may be either regulated or constitutive.

Structural genes, abundantly transcribed at late times in a viral infection cycle, provide particularly useful promoter sequences. Examples include sequences derived from the gene encoding the viral polyhedron protein, Friesen et al., (1986) “The Regulation of Baculovirus Gene Expression,” in: The Molecular Biology of Baculoviruses (ed. Walter Doerfler); EPO Publ. Nos. 127 839 and 155 476; and the gene encoding the p10 protein, Vlak et al., (1988), J. Gen. Virol. 69:765.

DNA encoding suitable signal sequences can be derived from genes for secreted insect or baculovirus proteins, such as the baculovirus polyhedrin gene (Carbonell et al. (1988) Gene, 73:409). Alternatively, since the signals for mammalian cell posttranslational modifications (such as signal peptide cleavage, proteolytic cleavage, and phosphorylation) appear to be recognized by insect cells, and the signals required for secretion and nuclear accumulation also appear to be conserved between the invertebrate cells and vertebrate cells, leaders of non-insect origin, such as those derived from genes encoding human α-interferon, Maeda et al., (1985), Nature 315:592; human gastrin-releasing peptide, Lebacq-Verheyden et al., (1988), Molec. Cell. Biol. 8:3129; human IL-2, Smith et al., (1985) Proc. Nat'l Acad. Sci. USA, 82:8404; mouse IL-3, (Miyajima et al., (1987) Gene 58:273; and human glucocerebrosidase, Martin et al. (1988) DNA, 7:99, can also be used to provide for secretion in insects.

A recombinant polypeptide or polyprotein may be expressed intracellularly or, if it is expressed with the proper regulatory sequences, it can be secreted. Good intracellular expression of nonfused foreign proteins usually requires heterologous genes that ideally have a short leader sequence containing suitable translation initiation signals preceding an ATG start signal. If desired, methionine at the N-terminus may be cleaved from the mature protein by in vitro incubation with cyanogen bromide.

Alternatively, recombinant polyproteins or proteins which are not naturally secreted can be secreted from the insect cell by creating chimeric DNA molecules that encode a fusion protein comprised of a leader sequence fragment that provides for secretion of the foreign protein in insects. The leader sequence fragment usually encodes a signal peptide comprised of hydrophobic amino acids which direct the translocation of the protein into the endoplasmic reticulum.

After insertion of the DNA sequence and/or the gene encoding the expression product precursor of the protein, an insect cell host is co-transformed with the heterologous DNA of the transfer vector and the genomic DNA of wild type baculovirus—usually by co-transfection. The promoter and transcription termination sequence of the construct will usually comprise a 2-5 kb section of the baculovirus genome. Methods for introducing heterologous DNA into the desired site in the baculovirus virus are known in the art (See Summers and Smith supra; Ju et al. (1987); Smith et al., Mol. Cell. Biol. (1983) 3:2156; and Luckow and Summers (1989)). For example, the insertion can be into a gene such as the polyhedrin gene, by homologous double crossover recombination; insertion can also be into a restriction enzyme site engineered into the desired baculovirus gene. Miller et al., (1989), Bioessays 4:91 The DNA sequence, when cloned in place of the polyhedrin gene in the expression vector, is flanked both 5′ and 3′ by polyhedrin-specific sequences and is positioned downstream of the polyhedrin promoter.

The newly formed baculovirus expression vector is subsequently packaged into an infectious recombinant baculovirus. Homologous recombination occurs at low frequency (between about 1% and about 5%); thus, the majority of the virus produced after cotransfection is still wild-type virus. Therefore, a method is necessary to identify recombinant viruses. An advantage of the expression system is a visual screen allowing recombinant viruses to be distinguished. The polyhedrin protein, which is produced by the native virus, is produced at very high levels in the nuclei of infected cells at late times after viral infection. Accumulated polyhedrin protein forms occlusion bodies that also contain embedded particles. These occlusion bodies, up to 15 μm in size, are highly refractile, giving them a bright shiny appearance that is readily visualized under the light microscope. Cells infected with recombinant viruses lack occlusion bodies. To distinguish recombinant virus from wild-type virus, the transfection supernatant is plaqued onto a monolayer of insect cells by techniques known to those skilled in the art. Namely, the plaques are screened under the light microscope for the presence (indicative of wild-type virus) or absence (indicative of recombinant virus) of occlusion bodies. “Current Protocols in Microbiology” Vol. 2 (Ausubel et al. eds) at 16.8 (Supp. 10, 1990); Summers and Smith, supra; Miller et al. (1989).

Recombinant baculovirus expression vectors have been developed for infection into several insect cells. For example, recombinant baculoviruses have been developed for, inter alia: Aedes aegypti, Autographa californica, Bombyx mori, Drosophila melanogaster, Spodoptera frugiperda, and Trichoplusia ni (WO 89/046699; Carbonell et al., (1985) J. Virol. 56:153; Wright (1986) Nature 321:718; Smith et al., (1983) Mol. Cell. Biol. 3:2156; and see generally, Fraser, et al. (1989) In Vitro Cell. Dev. Biol. 25:225).

Cells and cell culture media are commercially available for both direct and fusion expression of heterologous polypeptides in a baculovirus/expression system; cell culture technology is generally known to those skilled in the art. See, eg. Summers and Smith supra.

The modified insect cells may then be grown in an appropriate nutrient medium, which allows for stable maintenance of the plasmid(s) present in the modified insect host. Where the expression product gene is under inducible control, the host may be grown to high density, and expression induced. Alternatively, where expression is constitutive, the product will be continuously expressed into the medium and the nutrient medium must be continuously circulated, while removing the product of interest and augmenting depleted nutrients. The product may be purified by such techniques as chromatography, eg. HPLC, affinity chromatography, ion exchange chromatography, etc.; electrophoresis; density gradient centrifugation; solvent extraction, or the like. As appropriate, the product may be further purified, as required, so as to remove substantially any insect proteins which are also secreted in the medium or result from lysis of insect cells, so as to provide a product which is at least substantially free of host debris, eg. proteins, lipids and polysaccharides.

In order to obtain protein expression, recombinant host cells derived from the transformants are incubated under conditions which allow expression of the recombinant protein encoding sequence. These conditions will vary, dependent upon the host cell selected. However, the conditions are readily ascertainable to those of ordinary skill in the art, based upon what is known in the art.

iii. Plant Systems

There are many plant cell culture and whole plant genetic expression systems known in the art. Exemplary plant cellular genetic expression systems include those described in patents, such as: U.S. Pat. No. 5,693,506; U.S. Pat. No. 5,659,122; and U.S. Pat. No. 5,608,143. Additional examples of genetic expression in plant cell culture has been described by Zenk, Phytochemistry 30:3861-3863 (1991). Descriptions of plant protein signal peptides may be found in addition to the references described above in Vaulcombe et al., Mol. Gen. Genet. 209:33-40 (1987); Chandler et al., Plant Molecular Biology 3:407-418 (1984); Rogers, J. Biol. Chem. 260:3731-3738 (1985); Rothstein et al., Gene 55:353-356 (1987); Whittier et al., Nucleic Acids Research 15:2515-2535 (1987); Wirsel et al., Molecular Microbiology 3:3-14 (1989); Yu et al., Gene 122:247-253 (1992). A description of the regulation of plant gene expression by the phytohormone, gibberellic acid and secreted enzymes induced by gibberellic acid can be found in R. L. Jones and J. MacMillin, Gibberellins: in: Advanced Plant Physiology, Malcolm B. Wilkins, ed., 1984 Pitman Publishing Limited, London, pp. 21-52. References that describe other metabolically-regulated genes: Sheen, Plant Cell, 2:1027-1038 (1990); Maas et al., EMBO J. 9:3447-3452 (1990); Benkel and Hickey, Proc. Natl. Acad. Sci 84:1337-1339 (1987)

Typically, using techniques known in the art, a desired polynucleotide sequence is inserted into an expression cassette comprising genetic regulatory elements designed for operation in plants. The expression cassette is inserted into a desired expression vector with companion sequences upstream and downstream from the expression cassette suitable for expression in a plant host. The companion sequences will be of plasmid or viral origin and provide necessary characteristics to the vector to permit the vectors to move DNA from an original cloning host, such as bacteria, to the desired plant host. The basic bacterial/plant vector construct will preferably provide a broad host range prokaryote replication origin; a prokaryote selectable marker, and, for Agrobacterium transformations, T DNA sequences for Agrobacterium-mediated transfer to plant chromosomes. Where the heterologous gene is not readily amenable to detection, the construct will preferably also have a selectable marker gene suitable for determining if a plant cell has been transformed. A general review of suitable markers, for example for the members of the grass family, is found in Wilmink and Dons, 1993, Plant Mol. Biol. Reptr, 11(2):165-185.

Sequences suitable for permitting integration of the heterologous sequence into the plant genome are also recommended. These might include transposon sequences and the like for homologous recombination as well as Ti sequences which permit random insertion of a heterologous expression cassette into a plant genome. Suitable prokaryote selectable markers include resistance toward antibiotics such as ampicillin or tetracycline. Other DNA sequences encoding additional functions may also be present in the vector, as is known in the art.

The nucleic acid molecules of the subject invention may be included into an expression cassette for expression of the protein(s) of interest. Usually, there will be only one expression cassette, although two or more are feasible. The recombinant expression cassette will contain in addition to the heterologous protein encoding sequence the following elements, a promoter region, plant 5′ untranslated sequences, initiation codon depending upon whether or not the structural gene comes equipped with one, and a transcription and translation termination sequence. Unique restriction enzyme sites at the 5′ and 3′ ends of the cassette allow for easy insertion into a pre-existing vector.

A heterologous coding sequence may be for any protein relating to the present invention. The sequence encoding the protein of interest will encode a signal peptide which allows processing and translocation of the protein, as appropriate, and will usually lack any sequence which might result in the binding of the desired protein of the invention to a membrane. Since, for the most part, the transcriptional initiation region will be for a gene which is expressed and translocated during germination, by employing the signal peptide which provides for translocation, one may also provide for translocation of the protein of interest. In this way, the protein(s) of interest will be translocated from the cells in which they are expressed and may be efficiently harvested. Typically secretion in seeds are across the aleurone or scutellar epithelium layer into the endosperm of the seed. While it is not required that the protein be secreted from the cells in which the protein is produced, this facilitates the isolation and purification of the recombinant protein.

Since the ultimate expression of the desired gene product will be in a eucaryotic cell it is desirable to determine whether any portion of the cloned gene contains sequences which will be processed out as introns by the host's splicosome machinery. If so, site-directed mutagenesis of the “intron” region may be conducted to prevent losing a portion of the genetic message as a false intron code, Reed and Maniatis, Cell 41:95-105, 1985.

The vector can be microinjected directly into plant cells by use of micropipettes to mechanically transfer the recombinant DNA. Crossway, Mol. Gen. Genet, 202:179-185, 1985. The genetic material may also be transferred into the plant cell by using polyethylene glycol, Krens, et al., Nature, 296, 72-74, 1982. Another method of introduction of nucleic acid segments is high velocity ballistic penetration by small particles with the nucleic acid either within the matrix of small beads or particles, or on the surface, Klein, et al., Nature, 327, 70-73, 1987 and Knudsen and Muller, 1991, Planta, 185:330-336 teaching particle bombardment of barley endosperm to create transgenic barley. Yet another method of introduction would be fusion of protoplasts with other entities, either minicells, cells, lysosomes or other fusible lipid-surfaced bodies, Fraley, et al., Proc. Natl. Acad. Sci. USA, 79, 1859-1863, 1982.

The vector may also be introduced into the plant cells by electroporation. (Fromm et al., Proc. Natl Acad. Sci. USA 82:5824, 1985). In this technique, plant protoplasts are electroporated in the presence of plasmids containing the gene construct. Electrical impulses of high field strength reversibly permeabilize biomembranes allowing the introduction of the plasmids. Electroporated plant protoplasts reform the cell wall, divide, and form plant callus.

All plants from which protoplasts can be isolated and cultured to give whole regenerated plants can be transformed by the present invention so that whole plants are recovered which contain the transferred gene. It is known that practically all plants can be regenerated from cultured cells or tissues, including but not limited to all major species of sugarcane, sugar beet, cotton, fruit and other trees, legumes and vegetables. Some suitable plants include, for example, species from the genera Fragaria, Lotus, Medicago, Onobrychis, Trifolium, Trigonella, Vigna, Citrus, Linum, Geranium, Manihot, Daucus, Arabidopsis, Brassica, Raphanus, Sinapis, Atropa, Capsicum, Datura, Hyoscyamus, Lycopersion, Nicotiana, Solanum, Petunia, Digitalis, Majorana, Cichorium, Helianthus, Lactuca, Bromus, Asparagus, Antirrhinum, Hererocallis, Nemesia, Pelargonium, Panicum, Pennisetum, Ranunculus, Senecio, Salpiglossis, Cucumis, Browaalia, Glycine, Lolium, Zea, Triticum, Sorghum, and Datura.

Means for regeneration vary from species to species of plants, but generally a suspension of transformed protoplasts containing copies of the heterologous gene is first provided. Callus tissue is formed and shoots may be induced from callus and subsequently rooted. Alternatively, embryo formation can be induced from the protoplast suspension. These embryos germinate as natural embryos to form plants. The culture media will generally contain various amino acids and hormones, such as auxin and cytokinins. It is also advantageous to add glutamic acid and proline to the medium, especially for such species as corn and alfalfa. Shoots and roots normally develop simultaneously. Efficient regeneration will depend on the medium, on the genotype, and on the history of the culture. If these three variables are controlled, then regeneration is fully reproducible and repeatable.

In some plant cell culture systems, the desired protein of the invention may be excreted or alternatively, the protein may be extracted from the whole plant. Where the desired protein of the invention is secreted into the medium, it may be collected. Alternatively, the embryos and embryoless-half seeds or other plant tissue may be mechanically disrupted to release any secreted protein between cells and tissues. The mixture may be suspended in a buffer solution to retrieve soluble proteins. Conventional protein isolation and purification methods will be then used to purify the recombinant protein. Parameters of time, temperature pH, oxygen, and volumes will be adjusted through routine methods to optimize expression and recovery of heterologous protein.

iv. Bacterial Systems

Bacterial expression techniques are known in the art. A bacterial promoter is any DNA sequence capable of binding bacterial RNA polymerase and initiating the downstream (3′) transcription of a coding sequence (eg. structural gene) into mRNA. A promoter will have a transcription initiation region which is usually placed proximal to the 5′ end of the coding sequence. This transcription initiation region usually includes an RNA polymerase binding site and a transcription initiation site. A bacterial promoter may also have a second domain called an operator, that may overlap an adjacent RNA polymerase binding site at which RNA synthesis begins. The operator permits negative regulated (inducible) transcription, as a gene repressor protein may bind the operator and thereby inhibit transcription of a specific gene. Constitutive expression may occur in the absence of negative regulatory elements, such as the operator. In addition, positive regulation may be achieved by a gene activator protein binding sequence, which, if present is usually proximal (5′) to the RNA polymerase binding sequence. An example of a gene activator protein is the catabolite activator protein (CAP), which helps initiate transcription of the lac operon in Escherichia coli (E. coli) [Raibaud et al. (1984) Annu. Rev. Genet. 18:173]. Regulated expression may therefore be either positive or negative, thereby either enhancing or reducing transcription.

Sequences encoding metabolic pathway enzymes provide particularly useful promoter sequences. Examples include promoter sequences derived from sugar metabolizing enzymes, such as galactose, lactose (lac) [Chang et al. (1977) Nature 198:1056], and maltose. Additional examples include promoter sequences derived from biosynthetic enzymes such as tryptophan (trp) [Goeddel et al. (1980) Nuc. Acids Res. 8:4057; Yelverton et al. (1981) Nucl. Acids Res. 9:731; U.S. Pat. No. 4,738,921; EP-A-0036776 and EP-A-0121775]. The g-laotamase (bla) promoter system [Weissmann (1981) “The cloning of interferon and other mistates.” In Interferon 3 (ed I. Gresser)], bacteriophage lambda PL [Shimatake et al. (1981) Nature 292:128] and T5 [U.S. Pat. No. 4,689,406] promoter systems also provide useful promoter sequences.

In addition, synthetic promoters which do not occur in nature also function as bacterial promoters. For example, transcription activation sequences of one bacterial or bacteriophage promoter may be joined with the operon sequences of another bacterial or bacteriophage promoter, creating a synthetic hybrid promoter [U.S. Pat. No. 4,551,433]. For example, the tac promoter is a hybrid trp-lac promoter comprised of both trp promoter and lac operon sequences that is regulated by the lac repressor [Amann et al. (1983) Gene 25:167; de Boer et al. (1983) Proc. Natl. Acad. Sci. 80:21]. Furthermore, a bacterial promoter can include naturally occurring promoters of non-bacterial origin that have the ability to bind bacterial RNA polymerase and initiate transcription. A naturally occurring promoter of non-bacterial origin can also be coupled with a compatible RNA polymerase to produce high levels of expression of some genes in prokaryotes. The bacteriophage T7 RNA polymerase/promoter system is an example of a coupled promoter system [Studier et al. (1986) J. Mol. Biol. 189:113; Tabor et al. (1985) Proc Natl. Acad. Sci. 82:1074]. In addition, a hybrid promoter can also be comprised of a bacteriophage promoter and an E. coli operator region (EPO-A-0 267 851).

In addition to a functioning promoter sequence, an efficient ribosome binding site is also useful for the expression of foreign genes in prokaryotes. In E. coli, the ribosome binding site is called the Shine-Dalgarno (SD) sequence and includes an initiation codon (ATG) and a sequence 3-9 nucleotides in length located 3-11 nucleotides upstream of the initiation codon [Shine et al. (1975) Nature 254:34). The SD sequence is thought to promote binding of mRNA to the ribosome by the pairing of bases between the SD sequence and the 3′ and of E. coli 16S rRNA [Steitz et al. (1979) “Genetic signals and nucleotide sequences in messenger RNA.” In Biological Regulation and Development: Gene Expression (ed. R. F. Goldberger)]. To express eukaryotic genes and prokaryotic genes with weak ribosome-binding site [Sambrook et al. (1989) “Expression of cloned genes in Escherichia coli.” In Molecular Cloning: A Laboratory Manual].

A DNA molecule may be expressed intracellularly. A promoter sequence may be directly linked with the DNA molecule, in which case the first amino acid at the N-terminus will always be a methionine, which is encoded by the ATG start codon. If desired, methionine at the N-terminus may be cleaved from the protein by in vitro incubation with cyanogen bromide or by either in vivo on in vitro incubation with a bacterial methionine N-terminal peptidase (EPO-A-0 219 237).

Fusion proteins provide an alternative to direct expression. Usually, a DNA sequence encoding the N-terminal portion of an endogenous bacterial protein, or other stable protein, is fused to the 5′ end of heterologous coding sequences. Upon expression, this construct will provide a fusion of the two amino acid sequences. For example, the bacteriophage lambda cell gene can be linked at the 5′ terminus of a foreign gene and expressed in bacteria The resulting fusion protein preferably retains a site for a processing enzyme (factor Xa) to cleave the bacteriophage protein from the foreign gene [Nagai et al. (1984) Nature 309:810]. Fusion proteins can also be made with sequences from the lacZ [Jia et al. (1987) Gene 60:197], trpE (Allen et al. (1987) J. Biotechnol. 5:93; Makoff et al. (1989) J. Gen. Microbiol. 135:11], and Chey [EP-A-0 324 647] genes. The DNA sequence at the junction of the two amino acid sequences may or may not encode a cleavable site. Another example is a ubiquitin fusion protein. Such a fusion protein is made with the ubiquitin region that preferably retains a site for a processing enzyme (eg. ubiquitin specific processing-protease) to cleave the ubiquitin from the foreign protein. Through this method, native foreign protein can be isolated [Miller et al. (1989) Bio/Technology 7:698].

Alternatively, foreign proteins can also be secreted from the cell by creating chimeric DNA molecules that encode a fusion protein comprised of a signal peptide sequence fragment that provides for secretion of the foreign protein in bacteria [U.S. Pat. No. 4,336,336]. The signal sequence fragment usually encodes a signal peptide comprised of hydrophobic amino acids which direct the secretion of the protein from the cell. The protein is either secreted into the growth media (gram-positive bacteria) or into the periplasmic space, located between the inner and outer membrane of the cell (gram-negative bacteria). Preferably there are processing sites, which can be cleaved either in vivo or in vitro encoded between the signal peptide fragment and the foreign gene.

DNA encoding suitable signal sequences can be derived from genes for secreted bacterial proteins, such as the E. coli outer membrane protein gene (ompA) [Masui et al. (1983), in: Experimental Manipulation of Gene Expression; Ghrayeb et al. (1984) EMBO J. 3:2437] and the E. coli alkaline phosphatase signal sequence (phoA) [Oka et al. (1985) Proc. Natl. Acad. Sci. 82:7212]. As an additional example, the signal sequence of the alpha-amylase gene from various Bacillus strains can be used to secrete heterologous proteins from B. subtilis [Palva et al. (1982) Proc. Natl. Acad. Sci. USA 79:5582; EP-A-0 244 042].

Usually, transcription termination sequences recognized by bacteria are regulatory regions located 3′ to the translation stop codon, and thus together with the promoter flank the coding sequence. These sequences direct the transcription of an mRNA which can be translated into the polypeptide encoded by the DNA. Transcription termination sequences frequently include DNA sequences of about 50 nucleotides capable of forming stem loop structures that aid in terminating transcription. Examples include transcription termination sequences derived from genes with strong promoters, such as the trip gene in E. coli as well as other biosynthetic genes.

Usually, the above described components, comprising a promoter, signal sequence (if desired), coding sequence of interest, and transcription termination sequence, are put together into expression constructs. Expression constructs are often maintained in a replicon, such as an extrachromosomal element (eg. plasmids) capable of stable maintenance in a host, such as bacteria The replicon will have a replication system, thus allowing it to be maintained in a prokaryotic host either for expression or for cloning and amplification. In addition, a replicon may be either a high or low copy number plasmid. A high copy number plasmid will generally have a copy number ranging from about 5 to about 200, and usually about 10 to about 150. A host containing a high copy number plasmid will preferably contain at least about 10, and more preferably at least about 20 plasmids. Either a high or low copy number vector may be selected, depending upon the effect of the vector and the foreign protein on the host.

Alternatively, the expression constructs can be integrated into the bacterial genome with an integrating vector. Integrating vectors usually contain at least one sequence homologous to the bacterial chromosome that allows the vector to integrate. Integrations appear to result from recombinations between homologous DNA in the vector and the bacterial chromosome. For example, integrating vectors constructed with DNA from various Bacillus strains integrate into the Bacillus chromosome (EP-A-0 127 328). Integrating vectors may also be comprised of bacteriophage or transposon sequences.

Usually, extrachromosomal and integrating expression constructs may contain selectable markers to allow for the selection of bacterial strains that have been transformed. Selectable markers can be expressed in the bacterial host and may include genes which render bacteria resistant to drugs such as ampicillin, chloramphenicol, erythromycin, kanamycin (neomycin), and tetracycline [Davies et al. (1978) Annu. Rev. Microbiol. 32:469]. Selectable markers may also include biosynthetic genes, such as those in the histidine, tryptophan, and leucine biosynthetic pathways.

Alternatively, some of the above described components can be put together in transformation vectors. Transformation vectors are usually comprised of a selectable market that is either maintained in a replicon or developed into an integrating vector, as described above.

Expression and transformation vectors, either extra-chromosomal replicons or integrating vectors, have been developed for transformation into many bacteria For example, expression vectors have been developed for, inter alia, the following bacteria: Bacillus subtilis [Palva et al. (1982) Proc. Natl. Acad. Sci. USA 79:5582; EP-A-0 036 259 and EP-A-0 063 953; WO 84/04541], Escherichia coli [Shimatake et al. (1981) Nature 292:128; Amann et al. (1985) Gene 40:183; Studier et al. (1986) J. Mol. Biol. 189:113; EP-A-0 036 776, EP-A-0 136 829 and EP-A-0 136 907], Streptococcus cremoris [Powell et al. (1988) Appl. Environ. Microbiol. 54:655]; Streptococcus lividans [Powell et al. (1988) Appl. Environ. Microbiol. 54:655], Streptomyces lividans [U.S. Pat. No. 4,745,056].

Methods of introducing exogenous DNA into bacterial hosts are well-known in the art, and usually include either the transformation of bacteria treated with CaCl₂ or other agents, such as divalent cations and DMSO. DNA can also be introduced into bacterial cells by electroporation. Transformation procedures usually vary with the bacterial species to be transformed. See eg. [Masson et al. (1989) FEMS Microbiol. Lett. 60:273; Palva et al. (1982) Proc. Natl. Acad. Sci USA 79:5582; EP-A-0 036 259 and EP-A-0 063 953; WO 84/04541, Bacillus], [Miller et al. (1988) Proc. Natl. Acad. Sci. 85:856; Wang et al. (1990) J. Bacteriol. 172:949, Campylobacter], [Cohen et al. (1973) Proc. Natl. Acad. Sci. 69:2110; Dower et al. (1988) Nucleic Acids Res. 16:6127; Kushner (1978) “An improved method for transformation of Escherichia coli with ColE1-derived plasmids. In Genetic Engineering: Proceedings of the International Symposium on Genetic Engineering (eds. H. W. Boyer and S. Nicosia); Mandel et al. (1970) J. Mol. Biol. 53:159; Taketo (1988) Biochim Biophys. Acta 949:318; Escherichia], [Chassy et al. (1987) FEMS Microbiol. Lett. 44:173 Lactobacillus]; [Fiedler et al. (1988) Anal. Biochem 170:38, Pseudomonas]; [Augustin et al. (1990) FEMS Microbiol. Lett. 66:203, Staphylococcus], [Barany et al. (1980) J. Bacteriol. 144:698; Harlander (1987) “Transformation of Streptococcus lactis by electroporation, in: Streptococcal Genetics (ed. J. Ferretti and R. Curtiss III); Perry et al. (1981) Infect Immun. 32:1295; Powell et al. (1988) Appl. Environ. Microbiol. 54:655; Somkuti et al. (1987) Proc. 4th Evr. Cong. Biotechnology 1:412, Streptococcus].

v. Yeast Expression

Yeast expression systems are also known to one of ordinary skill in the art. A yeast promoter is any DNA sequence capable of binding yeast RNA polymerase and initiating the downstream (3′) transcription of a coding sequence (eg. structural gene) into mRNA. A promoter will have a transcription initiation region which is usually placed proximal to the 5′ end of the coding sequence. This transcription initiation region usually includes an RNA polymerase binding site (the “TATA Box”) and a transcription initiation site. A yeast promoter may also have a second domain called an upstream activator sequence (UAS), which, if present, is usually distal to the structural gene. The UAS permits regulated (inducible) expression. Constitutive expression occurs in the absence of a UAS. Regulated expression may be either positive or negative, thereby either enhancing or reducing transcription.

Yeast is a fermenting organism with an active metabolic pathway, therefore sequences encoding enzymes in the metabolic pathway provide particularly useful promoter sequences. Examples include alcohol dehydrogenase (ADH) (EP-A-0 284 044), enolase, glucokinase, glucose-6-phosphate isomerase, glyceraldehyde-3-phosphate-dehydrogenase (GAP or GAPDH), hexokinase, phosphofructokinase, 3-phosphoglycerate mutase, and pyruvate kinase (PyK) (EPO-A-0 329 203). The yeast PHO5 gene, encoding acid phosphatase, also provides useful promoter sequences [Myanohara et al. (1983) Proc. Natl. Acad. Sci. USA 80:1].

In addition, synthetic promoters which do not occur in nature also function as yeast promoters. For example, UAS sequences of one yeast promoter may be joined with the transcription activation region of another yeast promoter, creating a synthetic hybrid promoter. Examples of such hybrid promoters include the ADH regulatory sequence linked to the GAP transcription activation region (U.S. Pat. Nos. 4,876,197 and 4,880,734). Other examples of hybrid promoters include promoters which consist of the regulatory sequences of either the ADH2, GAL4, GAL10, OR PHO5 genes, combined with the transcriptional activation region of a glycolytic enzyme gene such as GAP or PyK (EP-A-0 164 556). Furthermore, a yeast promoter can include naturally occurring promoters of non-yeast origin that have the ability to bind yeast RNA polymerase and initiate transcription. Examples of such promoters include, inter alia, [Cohen et al. (1980) Proc. Natl. Acad. Sci. USA 77:1078; Henikoff et al. (1981) Nature 283:835; Hollenberg et al. (1981) Curr. Topics Microbiol. Immunol. 96:119; Hollenberg et al. (1979) “The Expression of Bacterial Antibiotic Resistance Genes in the Yeast Saccharomyces cerevisiae,” in: Plasmids of Medical, Environmental and Commercial Importance (eds. K. N. Timmis and A. Puhler); Mercerau-Puigalon et al. (1980) Gene 11:163; Panthier et al. (1980) Curr. Genet. 2:109;].

A DNA molecule may be expressed intracellularly in yeast. A promoter sequence may be directly linked with the DNA molecule, in which case the first amino acid at the N-terminus of the recombinant protein will always be a methionine, which is encoded by the ATG start codon. If desired, methionine at the N-terminus may be cleaved from the protein by in vitro incubation with cyanogen bromide.

Fusion proteins provide an alternative for yeast expression systems, as well as in mammalian, baculovirus, and bacterial expression systems. Usually, a DNA sequence encoding the N-terminal portion of an endogenous yeast protein, or other stable protein, is fused to the 5′ end of heterologous coding sequences. Upon expression, this construct will provide a fusion of the two amino acid sequences. For example, the yeast or human superoxide dismutase (SOD) gene, can be linked at the 5′ terminus of a foreign gene and expressed in yeast. The DNA sequence at the junction of the two amino acid sequences may or may not encode a cleavable site. See eg. EP-A-0 196 056. Another example is a ubiquitin fusion protein. Such a fusion protein is made with the ubiquitin region that preferably retains a site for a processing enzyme (eg. ubiquitin-specific processing protease) to cleave the ubiquitin from the foreign protein. Through this method, therefore, native foreign protein can be isolated (eg. WO88/024066).

Alternatively, foreign proteins can also be secreted from the cell into the growth media by creating chimeric DNA molecules that encode a fusion protein comprised of a leader sequence fragment that provide for secretion in yeast of the foreign protein. Preferably, there are processing sites encoded between the leader fragment and the foreign gene that can be cleaved either in vivo or in vitro. The leader sequence fragment usually encodes a signal peptide comprised of hydrophobic amino acids which direct the secretion of the protein from the cell.

DNA encoding suitable signal sequences can be derived from genes for secreted yeast proteins, such as the yeast invertase gene (EP-A-0 012 873; JPO. 62,096,086) and the A-factor gene (U.S. Pat. No. 4,588,684). Alternatively, leaders of non-yeast origin, such as an interferon leader, exist that also provide for secretion in yeast (EP-A-0 060 057).

A preferred class of secretion leaders are those that employ a fragment of the yeast alpha-factor gene, which contains both a “pre” signal sequence, and a “pro” region. The types of alpha-factor fragments that can be employed include the full-length pre-pro alpha factor leader (about 83 amino acid residues) as well as truncated alpha-factor leaders (usually about 25 to about 50 amino acid residues) (U.S. Pat. Nos. 4,546,083 and 4,870,008; EP-A-0 324 274). Additional leaders employing an alpha-factor leader fragment that provides for secretion include hybrid alpha-factor leaders made with a presequence of a first yeast, but a pro-region from a second yeast alphafactor. (eg. see WO 89/02463.)

Usually, transcription termination sequences recognized by yeast are regulatory regions located 3′ to the translation stop codon, and thus together with the promoter flank the coding sequence. These sequences direct the transcription of an mRNA which can be translated into the polypeptide encoded by the DNA. Examples of transcription terminator sequence and other yeast-recognized termination sequences, such as those coding for glycolytic enzymes.

Usually, the above described components, comprising a promoter, leader (if desired), coding sequence of interest, and transcription termination sequence, are put together into expression constructs. Expression constructs are often maintained in a replicon, such as an extrachromosomal element (eg. plasmids) capable of stable maintenance in a host, such as yeast or bacteria. The replicon may have two replication systems, thus allowing it to be maintained, for example, in yeast for expression and in a prokaryotic host for cloning and amplification. Examples of such yeast-bacteria shuttle vectors include YEp24 [Botstein et al. (1979) Gene 8:17-24], pCl/1 [Brake et al. (1984) Proc. Natl. Acad. Sci USA 81:4642-4646], and YRp17 [Stinchcomb et al. (1982) J. Mol. Biol. 158:157]. In addition, a replicon may be either a high or low copy number plasmid. A high copy number plasmid will generally have a copy number ranging from about 5 to about 200, and usually about 10 to about 150. A host containing a high copy number plasmid will preferably have at least about 10, and more preferably at least about 20. Enter a high or low copy number vector may be selected, depending upon the effect of the vector and the foreign protein on the host. See eg. Brake et al., supra.

Alternatively, the expression constructs can be integrated into the yeast genome with an integrating vector. Integrating vectors usually contain at least one sequence homologous to a yeast chromosome that allows the vector to integrate, and preferably contain two homologous sequences flanking the expression construct. Integrations appear to result from recombinations between homologous DNA in the vector and the yeast chromosome [Orr-Weaver et al. (1983) Methods in Enzymol. 101:228-245]. An integrating vector may be directed to a specific locus in yeast by selecting the appropriate homologous sequence for inclusion in the vector. See Orr-Weaver et al., supra. One or more expression construct may integrate, possibly affecting levels of recombinant protein produced [Rine et al. (1983) Proc. Natl. Acad. Sci. USA 80:6750]. The chromosomal sequences included in the vector can occur either as a single segment in the vector, which results in the integration of the entire vector, or two segments homologous to adjacent segments in the chromosome and flanking the expression construct in the vector, which can result in the stable integration of only the expression construct.

Usually, extrachromosomal and integrating expression constructs may contain selectable markers to allow for the selection of yeast strains that have been transformed. Selectable markers may include biosynthetic genes that can be expressed in the yeast host, such as ADE2, HIS4, LEU2, TRP1, and ALG7, and the G418 resistance gene, which confer resistance in yeast cells to tunicamycin and G418, respectively. In addition, a suitable selectable marker may also provide yeast with the ability to grow in the presence of toxic compounds, such as metal. For example, the presence of CUP1 allows yeast to grow in the presence of copper ions [Butt et al. (1987) Microbiol, Rev. 51:351].

Alternatively, some of the above described components can be put together into transformation vectors. Transformation vectors are usually comprised of a selectable marker that is either maintained in a replicon or developed into an integrating vector, as described above.

Expression and transformation vectors, either extrachromosomal replicons or integrating vectors, have been developed for transformation into many yeasts. For example, expression vectors have been developed for, inter alia, the following yeasts: Candida albicans [Kurtz, et al. (1986) Mol. Cell. Biol. 6:142], Candida maltosa [Kunze, et al. (1985) J. Basic Microbiol. 25:141]. Hansenula polymorpha [Gleeson, et al. (1986) J. Gen. Microbiol. 132:3459; Roggenkamp et al. (1986) Mol. Gen. Genet. 202:302], Kluyveromyces fragilis [Das, et al. (1984) J. Bacteriol. 158:1165], Kluyveromyces lactis [De Louvencourt et al. (1983) J. Bacteriol. 154:737; Van den Berg et al. (1990) Bio/Technology 8:135], Pichia guillerimondii [Kunze et al. (1985) J. Basic Microbiol. 25:141], Pichia pastoris [Cregg, et al. (1985) Mol. Cell. Biol. 5:3376; U.S. Pat. Nos. 4,837,148 and 4,929,555], Saccharomyces cerevisiae [Hinnen et al. (1978) Proc. Natl. Acad. Sci. USA 75:1929; Ito et al. (1983) J. Bacteriol. 153:163], Schizosaccharomyces pombe [Beach and Nurse (1981) Nature 300:706], and Yarrowia lipolytica [Davidow, et al. (1985) Curr. Genet. 10:380471 Gaillardin, et al (1985) Curr. Genet. 10:49].

Methods of introducing exogenous DNA into yeast hosts are well-known in the art, and usually include either the transformation of spheroplasts or of intact yeast cells treated with alkali cations. Transformation procedures usually vary with the yeast species to be transformed. See eg. [Kurtz et al. (1986) Mol. Cell. Biol. 6:142; Kunze et al. (1985) J. Basic Microbiol. 25:141; Candida]; [Gleeson et al. (1986) J. Gen. Microbiol. 132:3459; Roggenkamp et al. (1986) Mol. Gen. Genet. 202:302; Hansenula]; [Das et al. (1984) J. Bacteriol. 158:1165; De Louvencourt et al. (1983) J. Bacteriol. 154:1165; Van den Berg et al. (1990) Bio/Technology 8:135; Kluyveromyces]; [Cregg et al. (1985) Mol. Cell. Biol. 5:3376; Kunze et al. (1985) J. Basic Microbiol. 25:141; U.S. Pat. Nos. 4,837,148 and 4,929,555; Pichia]; [Hinnen et al. (1978) Proc. Natl. Acad. Sci. USA 75;1929; Ito et al. (1983) J. Bacteriol. 153:163 Saccharomyces]; [Beach and Nurse (1981) Nature 300:706; Schizosaccharomyces]; [Davidow et al. (1985) Curr. Genet. 10:39; Gaillardin et al. (1985) Curr. Genet. 10:49; Yarrowia].

Antibodies

As used herein, the term “antibody” refers to a polypeptide or group of polypeptides composed of at least one antibody combining site. An “antibody combining site” is the three-dimensional binding space with an internal surface shape and charge distribution complementary to the features of an epitope of an antigen, which allows a binding of the antibody with the antigen. “Antibody” includes, for example, vertebrate antibodies, hybrid antibodies, chimeric antibodies, humanised antibodies, altered antibodies, univalent antibodies, Fab proteins, and single domain antibodies. Antibodies against the proteins of the invention are useful for affinity chromatography, immunoassays, and distinguishing/identifying Neisserial proteins.

Antibodies to the proteins of the invention, both polyclonal and monoclonal, may be prepared by conventional methods. In general, the protein is first used to immunize a suitable animal, preferably a mouse, rat, rabbit or goat. Rabbits and goats are preferred for the preparation of polyclonal sera due to the volume of serum obtainable, and the availability of labeled anti-rabbit and anti-goat antibodies. Immunization is generally performed by mixing or emulsifying the protein in saline, preferably in an adjuvant such as Freund's complete adjuvant, and injecting the mixture or emulsion parenterally (generally subcutaneously or intramuscularly). A dose of 50-200 μg/injection is typically sufficient. Immunization is generally boosted 2-6 weeks later with one or more injections of the protein in saline, preferably using Freund's incomplete adjuvant. One may alternatively generate antibodies by in vitro immunization using methods known in the adt, which for the purposes of this invention is considered equivalent to in vivo immunization. Polyclonal antisera is obtained by bleeding the immunized animal into a glass or plastic container, incubating the blood at 25° C. for one hour, followed by incubating at 4° C. for 2-18 hours. The serum is recovered by centrifugation (eg. 1,000 g for 10 minutes). About 20-50 ml per bleed may be obtained from rabbits.

Monoclonal antibodies are prepared using the standard method of Kohler & Milstein [Nature (1975) 256:495-96], or a modification thereof. Typically, a mouse or rat is immunized as described above. However, rather than bleeding the animal to extract serum, the spleen (and optionally several large lymph nodes) is removed and dissociated into single cells. If desired, the spleen cells may be screened (after removal of nonspecifically adherent cells) by applying a cell suspension to a plate or well coated with the protein antigen. B-cells expressing membrane-bound immunoglobulin specific for the antigen bind to the plate, and are not rinsed away with the rest of the suspension. Resulting B-cells, or all dissociated spleen cells, are then induced to fuse with myeloma cells to form hybridomas, and are cultured in a selective medium (eg. hypoxanthine, aminopterin, thymidine medium, “HAT”). The resulting hybridomas are plated by limiting dilution, and are assayed for the production of antibodies which bind specifically to the immunizing antigen (and which do not bind to unrelated antigens). The selected MAb-secreting hybridomas are then cultured either in vitro (eg. in tissue culture bottles or hollow fiber reactors), or in vivo (as ascites in mice).

If desired, the antibodies (whether polyclonal or monoclonal) may be labeled using conventional techniques. Suitable labels include fluorophores, chromophores, radioactive atoms (particularly ³²P and ¹²⁵I), electron-dense reagents, enzymes, and ligands having specific binding partners. Enzymes are typically detected by their activity. For example, horseradish peroxidase is usually detected by its ability to convert 3,3′,5,5′-tetramethylbenzidine (TMB) to a blue pigment, quantifiable with a spectrophotometer. “Specific binding partner” refers to a protein capable of binding a ligand molecule with high specificity, as for example in the case of an antigen and a monoclonal antibody specific therefor. Other specific binding partners include biotin and avidin or streptavidin, IgG and protein A, and the numerous receptor-ligand couples known in the art. It should be understood that the above description is not meant to categorize the various labels into distinct classes, as the same label may serve in several different modes. For example, ¹²⁵I may serve as a radioactive label or as an electron-dense reagent. HRP may serve as enzyme or as antigen for a MAb. Further, one may combine various labels for desired effect. For example, MAbs and avidin also require labels in the practice of this invention: thus, one might label a MAb with biotin, and detect its presence with avidin labeled with ¹²⁵I, or with an anti-biotin MAb labeled with HRP. Other permutations and possibilities will be readily apparent to those of ordinary skill in the art, and are considered as equivalents within the scope of the instant invention.

Pharmaceutical Compositions

Pharmaceutical compositions can comprise either polypeptides, antibodies, or nucleic acid of the invention. The pharmaceutical compositions will comprise a therapeutically effective amount of either polypeptides, antibodies, or polynucleotides of the claimed invention.

The term “therapeutically effective amount” as used herein refers to an amount of a therapeutic agent to treat, ameliorate, or prevent a desired disease or condition, or to exhibit a detectable therapeutic or preventative effect. The effect can be detected by, for example, chemical markers or antigen levels. Therapeutic effects also include reduction in physical symptoms, such as decreased body temperature. The precise effective amount for a subject will depend upon the subject's size and health, the nature and extent of the condition, and the therapeutics or combination of therapeutics selected for administration. Thus, it is not useful to specify an exact effective amount in advance. However, the effective amount for a given situation can be determined by routine experimentation and is within the judgement of the clinician.

For purposes of the present invention, an effective dose will be from about 0.01 mg/kg to 50 mg/kg or 0.05 mg/kg to about 10 mg/kg of the DNA constructs in the individual to which it is administered.

A pharmaceutical composition can also contain a pharmaceutically acceptable carrier. The term “pharmaceutically acceptable carrier” refers to a carrier for administration of a therapeutic agent, such as antibodies or a polypeptide, genes, and other therapeutic agents. The term refers to any pharmaceutical carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition, and which may be administered without undue toxicity. Suitable carriers may be large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, and inactive virus particles. Such carriers are well known to those of ordinary skill in the art.

Pharmaceutically acceptable salts can be used therein, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like. A thorough discussion of pharmaceutically acceptable excipients is available in Remington's Pharmaceutical Sciences (Mack Pub. Co., N.J. 1991).

Pharmaceutically acceptable carriers in therapeutic compositions may contain liquids such as water, saline, glycerol and ethanol. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles. Typically, the therapeutic compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection may also be prepared. Liposomes are included within the definition of a pharmaceutically acceptable carrier.

Delivery Methods

Once formulated, the compositions of the invention can be administered directly to the subject. The subjects to be treated can be animals; in particular, human subjects can be treated.

Direct delivery of the compositions will generally be accomplished by injection, either subcutaneously, intraperitoneally, intravenously or intramuscularly or delivered to the interstitial space of a tissue. The compositions can also be administered into a lesion. Other modes of administration include oral and pulmonary administration, suppositories, and transdermal or transcutaneous applications (eg. see WO98/20734), needles, and gene guns or hyposprays. Dosage treatment may be a single dose schedule or a multiple dose schedule.

Vaccines

Vaccines according to the invention may either be prophylactic (ie. to prevent infection) or therapeutic (ie. to treat disease after infection).

Such vaccines comprise immunising antigen(s), immunogen(s), polypeptide(s), protein(s) or nucleic acid, usually in combination with “pharmaceutically acceptable carriers,” which include any carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition. Suitable carriers are typically large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, lipid aggregates (such as oil droplets or liposomes), and inactive virus particles. Such carriers are well known to those of ordinary skill in the art. Additionally, these carriers may function as immunostimulating agents (“adjuvants”). Furthermore, the antigen or immunogen may be conjugated to a bacterial toxoid, such as a toxoid from diphtheria, tetanus, cholera, H. pylori, etc. pathogens.

Preferred adjuvants to enhance effectiveness of the composition include, but are not limited to: (1) aluminum salts (alum), such as aluminum hydroxide, aluminum phosphate, aluminum sulfate, etc; (2) oil-in-water emulsion formulations (with or without other specific immunostimulating agents such as muramyl peptides (see below) or bacterial cell wall components), such as for example (a) MF59™ (WO 90/14837; Chapter 10 in Vaccine design: the subunit and adjuvant approach, eds. Powell & Newman, Plenum Press 1995), containing 5% Squalene, 0.5% Tween 80, and 0.5% Span 85 (optionally containing various amounts of MTP-PE (see below), although not required) formulated into submicron particles using a microfluidizer such as Model 110Y microfluidizer (Microfluidics, Newton, Mass.), (b) SAF, containing 10% Squalane, 0.4% Tween 80, 5% pluronic-blocked polymer L121, and thr-MDP (see below) either microfluidized into a submicron emulsion or vortexed to generate a larger particle size emulsion, and (c) Ribi™ adjuvant system (RAS), (Ribi Immunochem, Hamilton, Mont.) containing 2% Squalene, 0.2% Tween 80, and one or more bacterial cell wall components from the group consisting of monophosphorylipid A (MPL), trehalose dimycolate (TDM), and cell wall skeleton (CWS), preferably MPL+CWS (Detox™); (3) saponin adjuvants, such as Stimulon™ (Cambridge Bioscience, Worcester, Mass.) may be used or particles generated therefrom such as ISCOMs (immunostimulating complexes); (4) Complete Freund's Adjuvant (CFA) and Incomplete Freund's Adjuvant (IFA); (5) cytokines, such as interleukins (eg. IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, etc.), interferons (eg. gamma interferon), macrophage colony stimulating factor (M-CSF), tumor necrosis factor (TNF), etc; and (6) other substances that act as immunostimulating agents to enhance the effectiveness of the composition. Alum and MF59™ are preferred.

As mentioned above, muramyl peptides include, but are not limited to, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)ethylamine (MTP-PE), etc.

The immunogenic compositions (eg. the immunising antigen/immunogen/polypeptide/protein/nucleic acid, pharmaceutically acceptable carrier, and adjuvant) typically will contain diluents, such as water, saline, glycerol, ethanol, etc. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles.

Typically, the immunogenic compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection may also be prepared. The preparation also may be emulsified or encapsulated in liposomes for enhanced adjuvant effect, as discussed above under pharmaceutically acceptable carriers.

Immunogenic compositions used as vaccines comprise an immunologically effective amount of the antigenic or immunogenic polypeptides, as well as any other of the above-mentioned components, as needed. By “immunologically effective amount”, it is meant that the administration of that amount to an individual, either in a single dose or as part of a series, is effective for treatment or prevention. This amount varies depending upon the health and physical condition of the individual to be treated, the taxonomic group of individual to be treated (eg. nonhuman primate, primate, etc.), the capacity of the individual's immune system to synthesize antibodies, the degree of protection desired, the formulation of the vaccine, the treating doctors assessment of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials.

The immunogenic compositions are conventionally administered parenterally, eg. by injection, either subcutaneously, intramuscularly, or transdermally/transcutaneously (eg. WO98/20734). Additional formulations suitable for other modes of administration include oral and pulmonary formulations, suppositories, and transdermal applications. Dosage treatment may be a single dose schedule or a multiple dose schedule. The vaccine may be administered in conjunction with other immunoregulatory agents.

As an alternative to protein-based vaccines, DNA vaccination may be employed [eg. Robinson & Torres (1997) Seminars in Immunology 9:271-283; Donnelly et al. (1997) Annu Rev Immunol 15:617-648; see later herein].

Gene Delivery Vehicles

Gene therapy vehicles for delivery of constructs including a coding sequence of a therapeutic of the invention, to be delivered to the mammal for expression in the mammal, can be administered either locally or systemically. These constructs can utilize viral or non-viral vector approaches in in vivo or ex vivo modality. Expression of such coding sequence can be induced using endogenous mammalian or heterologous promoters. Expression of the coding sequence in vivo can be either constitutive or regulated.

The invention includes gene delivery vehicles capable of expressing the contemplated nucleic acid sequences. The gene delivery vehicle is preferably a viral vector and, more preferably, a retroviral, adenoviral, adeno-associated viral (AAV), herpes viral, or alphavirus vector. The viral vector can also be an astrovirus, coronavirus, orthomyxovirus, papovavirus, paramyxovirus, parvovirus, picomavirus, poxvirus, or togavirus viral vector. See generally, Jolly (1994) Cancer Gene Therapy 1:51-64; Kimura (1994) Human Gene Therapy 5:845-852; Connelly (1995) Human Gene Therapy 6:185-193; and Kaplitt (1994) Nature Genetics 6:148-153.

Retroviral vectors are well known in the art and we contemplate that any retroviral gene therapy vector is employable in the invention, including B, C and D type retroviruses, xenotropic retroviruses (for example, NZB-X1, NZB-X2 and NZB9-1 (see O'Neill (1985) J. Virol. 53:160) polytropic retrovirus eg. MCF and MCF-MLV (see Kelly (1983) J. Virol. 45:291), spumaviruses and lentiviruses. See RNA Tumor Viruses, Second Edition, Cold Spring Harbor Laboratory, 1985.

Portions of the retroviral gene therapy vector may be derived from different retroviruses. For example, retrovector LTRs may be derived from a Murine Sarcoma Virus, a tRNA binding site from a Rous Sarcoma Virus, a packaging signal from a Murine Leukemia Virus, and an origin of second strand synthesis from an Avian Leukosis Virus.

These recombinant retroviral vectors may be used to generate transduction competent retroviral vector particles by introducing them into appropriate packaging cell lines (see U.S. Pat. No. 5,591,624). Retrovirus vectors can be constructed for site-specific integration into host cell DNA by incorporation of a chimeric integrase enzyme into the retroviral particle (see WO96/37626). It is preferable that the recombinant viral vector is a replication defective recombinant virus.

Packaging cell lines suitable for use with the above-described retrovirus vectors are well known in the art, are readily prepared (see WO95/30763 and WO92/05266), and can be used to create producer cell lines (also termed vector cell lines or “VCLs”) for the production of recombinant vector particles. Preferably, the packaging cell lines are made from human parent cells (eg. HT1080 cells) or mink parent cell lines, which eliminates inactivation in human serum.

Preferred retroviruses for the construction of retroviral gene therapy vectors include Avian Leukosis Virus, Bovine Leukemia, Virus, Murine Leukemia Virus, Mink-Cell Focus-Inducing Virus, Murine Sarcoma Virus, Reticuloendotheliosis Virus and Rous Sarcoma Virus. Particularly preferred Murine Leukemia Viruses include 4070A and 1504A (Hartley and Rowe (1976) J Virol 19:19-25), Abelson (ATCC No. VR-999), Friend (ATCC No. VR-245), Graffi, Gross (ATCC Nol VR-590), Kirsten, Harvey Sarcoma Virus and Rauscher (ATCC No. VR-998) and Moloney Murine Leukemia Virus (ATCC No. VR-190). Such retroviruses may be obtained from depositories or collections such as the American Type Culture Collection (“ATCC”) in Rockville, Md. or isolated from known sources using commonly available techniques.

Exemplary known retroviral gene therapy vectors employable in this invention include those described in patent applications GB2200651, EP0415731, EP0345242, EP0334301, WO89/02468; WO89/05349, WO89/09271, WO90/02806, WO90/07936, WO94/03622, WO93/25698, WO93/25234, WO93/11230, WO93/10218, WO91/02805, WO91/02825, WO95/07994, U.S. Pat. No. 5,219,740, U.S. Pat. No. 4,405,712, U.S. Pat. No. 4,861,719, U.S. Pat. No. 4,980,289, U.S. Pat. No. 4,777,127, U.S. Pat. No. 5,591,624. See also Vile (1993) Cancer Res 53:3860-3864; Vile (1993) Cancer Res 53:962-967; Rain (1993) Cancer Res 53 (1993) 83-88; Takamiya (1992) J Neurosci Res 33:493-503; Baba (1993) J Neurosurg 79:729-735; Mann (1983) Cell 33:153; Cane (1984) Proc Natl Acad Sci 81:6349; and Miller (1990) Human Gene Therapy 1.

Human adenoviral gene therapy vectors are also known in the art and employable in this invention. See, for example, Berkner (1988) Biotechniques 6:616 and Rosenfeld (1991) Science 252:431, and WO93/07283, WO93/06223, and WO93/07282. Exemplary known adenoviral gene therapy vectors employable in this invention include those described in the above referenced documents and in WO94/12649, WO93/03769, WO93/19191, WO94/28938, WO95/11984, WO95/00655, WO95/27071, WO95/29993, WO95/34671, WO96/05320, WO94/08026, WO94/11506, WO93/06223, WO94/24299, WO95/14102, WO95/24297, WO95/02697, WO94/28152, WO94/24299, WO95/09241, WO95/25807, WO95/05835, WO94/18922 and WO95/09654. Alternatively, administration of DNA linked to killed adenovirus as described in Curiel (1992) Hum. Gene Ther. 3:147-154 may be employed. The gene delivery vehicles of the invention also include adenovirus associated virus (AAV) vectors. Leading and preferred examples of such vectors for use in this invention are the AAV-2 based vectors disclosed in Srivastava, WO93/09239. Most preferred AAV vectors comprise the two AAV inverted terminal repeats in which the native D-sequences are modified by substitution of nucleotides, such that at least 5 native nucleotides and up to 18 native nucleotides, preferably at least 10 native nucleotides up to 18 native nucleotides, most preferably 10 native nucleotides are retained and the remaining nucleotides of the D-sequence are deleted or replaced with non-native nucleotides. The native D-sequences of the AAV inverted terminal repeats are sequences of 20 consecutive nucleotides in each AAV inverted terminal repeat (ie. there is one sequence at each end) which are not involved in HP formation. The non-native replacement nucleotide may be any nucleotide other than the nucleotide found in the native D-sequence in the same position. Other employable exemplary AAV vectors are pWP-19, pWN-1, both of which are disclosed in Nahreini (1993) Gene 124:257-262. Another example of such an AAV vector is psub201 (see Samulski (1987) J. Virol. 61:3096). Another exemplary AAV vector is the Double-D ITR vector. Construction of the Double-D ITR vector is disclosed in U.S. Pat. No. 5,478,745. Still other vectors are those disclosed in Carter U.S. Pat. No. 4,797,368 and Muzyczka U.S. Pat. No. 5,139,941, Chartejee U.S. Pat. No. 5,474,935, and Kotin WO94/288157. Yet a further example of an AAV vector employable in this invention is SSV9AFABTKneo, which contains the AFP enhancer and albumin promoter and directs expression predominantly in the liver. Its structure and construction are disclosed in Su (1996) Human Gene Therapy 7:463-470. Additional AAV gene therapy vectors are described in U.S. Pat. No. 5,354,678, U.S. Pat. No. 5,173,414, U.S. Pat. No. 5,139,941, and U.S. Pat. No. 5,252,479.

The gene therapy vectors of the invention also include herpes vectors. Leading and preferred examples are herpes simplex virus vectors containing a sequence encoding a thymidine kinase polypeptide such as those disclosed in U.S. Pat. No. 5,288,641 and EP0176170 (Roizman). Additional exemplary herpes simplex virus vectors include HFEM/ICP6-LacZ disclosed in WO95/04139 (Wistar Institute), pHSVlac described in Geller (1988) Science 241:1667-1669 and in WO90/09441 and WO92/07945, HSV Us3::pgC-lacZ described in Fink (1992) Human Gene Therapy 3:11-19 and HSV 7134, 2 RH 105 and GAL4 described in EP 0453242 (Breakefield), and those deposited with the ATCC as accession numbers ATCC VR-977 and ATCC VR-260.

Also contemplated are alpha virus gene therapy vectors that can be employed in this invention. Preferred alpha virus vectors are Sindbis viruses vectors. Togaviruses, Semliki Forest virus (ATCC VR-67; ATCC VR-1247), Middleberg virus (ATCC VR-370), Ross River virus (ATCC VR-373; ATCC VR-1246), Venezuelan equine encephalitis virus (ATCC VR923; ATCC VR-1250; ATCC VR-1249; ATCC VR-532), and those described in U.S. Pat. Nos. 5,091,309, 5,217,879, and WO92/10578. More particularly, those alpha virus vectors described in U.S. Ser. No. 08/405,627, filed Mar. 15, 1995, WO94/21792, WO92/10578, WO95/07994, U.S. Pat. No. 5,091,309 and U.S. Pat. No. 5,217,879 are employable. Such alpha viruses may be obtained from depositories or collections such as the ATCC in Rockville, Md. or isolated from known sources using commonly available techniques. Preferably, alphavirus vectors with reduced cytotoxicity are used (see U.S. Ser. No. 08/679,640).

DNA vector systems such as eukaryotic layered expression systems are also useful for expressing the nucleic acids of the invention. See WO95/07994 for a detailed description of eukaryotic layered expression systems. Preferably, the eukaryotic layered expression systems of the invention are derived from alphavirus vectors and most preferably from Sindbis viral vectors.

Other viral vectors suitable for use in the present invention include those derived from poliovirus, for example ATCC VR-58 and those described in Evans, Nature 339 (1989) 385 and Sabin (1973) J. Biol. Standardization 1:115; rhinovirus, for example ATCC VR-1110 and those described in Arnold (1990) J Cell Biochem L401; pox viruses such as canary pox virus or vaccinia virus, for example ATCC VR-111 and ATCC VR-2010 and those described in Fisher-Hoch (1989) Proc Natl Acad Sci 86:317; Flexner (1989) Ann NY Acad Sci 569:86, Flexner (1990) Vaccine 8:17; in U.S. Pat. No. 4,603,112 and U.S. Pat. No. 4,769,330 and WO89/01973; SV40 virus, for example ATCC VR-305 and those described in Mulligan (1979) Nature 277:108 and Madzak (1992) J Gen Virol 73:1533; influenza virus, for example ATCC VR-797 and recombinant influenza viruses made employing reverse genetics techniques as described in U.S. Pat. No. 5,166,057 and in Enami (1990) Proc Nail Acad Sci 87:3802-3805; Enami & Palese (1991) J Virol 65:2711-2713 and Luytjes (1989) Cell 59:110, (see also McMichael (1983) NEJ Med 309:13, and Yap (1978) Nature 273:238 and Nature (1979) 277:108); human immunodeficiency virus as described in EP-0386882 and in Buchschacher (1992) J. Virol. 66:2731; measles virus, for example ATCC VR-67 and VR-1247 and those described in EP-0440219; Aura virus, for example ATCC VR-368; Bebaru virus, for example ATCC VR-600 and ATCC VR-1240; Cabassou virus, for example ATCC VR-922; Chikungunya virus, for example ATCC VR-64 and ATCC VR-1241; Fort Morgan Virus, for example ATCC VR-924; Getah virus, for example ATCC VR-369 and ATCC VR-1243; Kyzylagach virus, for example ATCC VR-927; Mayaro virus, for example ATCC VR-66; Mucambo virus, for example ATCC VR-580 and ATCC VR-1244; Ndumu virus, for example ATCC VR-371; Pixuna virus, for example ATCC VR-372 and ATCC VR-1245; Tonate virus, for example ATCC VR-925; Triniti virus, for example ATCC VR-469; Una virus, for example ATCC VR-374; Whataroa virus, for example ATCC VR-926; Y-62-33 virus, for example ATCC VR-375; O'Nyong virus, Eastern encephalitis virus, for example ATCC VR-65 and ATCC VR-1242; Western encephalitis virus, for example ATCC VR-70, ATCC VR-1251, ATCC VR-622 and ATCC VR-1252; and coronavirus, for example ATCC VR-740 and those described in Hamre (1966) Proc Soc Exp Biol Med 121:190.

Delivery of the compositions of this invention into cells is not limited to the above mentioned viral vectors. Other delivery methods and media may be employed such as, for example, nucleic acid expression vectors, polycationic condensed DNA linked or unlinked to killed adenovirus alone, for example see U.S. Ser. No. 08/366,787, filed Dec. 30, 1994 and Curiel (1992) Hum Gene Ther 3:147-154 ligand linked DNA, for example see Wu (1989) J Biol Chem 264:16985-16987, eucaryotic cell delivery vehicles cells, for example see U.S. Ser. No. 08/240,030, filed May 9, 1994, and U.S. Ser. No. 08/404,796, deposition of photopolymerized hydrogel materials, hand-held gene transfer particle gun, as described in U.S. Pat. No. 5,149,655, ionizing radiation as described in U.S. Pat. No. 5,206,152 and in WO92/11033, nucleic charge neutralization or fusion with cell membranes. Additional approaches are described in Philip (1994) Mol Cell Biol 14:2411-2418 and in Woffendin (1994) Proc Natl Acad Sci 91:1581-1585.

Particle mediated gene transfer may be employed, for example see U.S. Ser. No. 60/023,867. Briefly, the sequence can be inserted into conventional vectors that contain conventional control sequences for high level expression, and then incubated with synthetic gene transfer molecules such as polymeric DNA-binding cations like polylysine, protamine, and albumin, linked to cell targeting ligands such as asialoorosomucoid, as described in Wu & Wu (1987) J. Biol. Chem. 262:4429-4432, insulin as described in Hucked (1990) Biochem Pharmacol 40:253-263, galactose as described in Plank (1992) Bioconjugate Chem 3:533-539, lactose or transferrin.

Naked DNA may also be employed. Exemplary naked DNA introduction methods are described in WO 90/11092 and U.S. Pat. No. 5,580,859. Uptake efficiency may be improved using biodegradable latex beads. DNA coated latex beads are efficiently transported into cells after endocytosis initiation by the beads. The method may be improved further by treatment of the beads to increase hydrophobicity and thereby facilitate disruption of the endosome and release of the DNA into the cytoplasm.

Liposomes that can act as gene delivery vehicles are described in U.S. Pat. No. 5,422,120, WO95/13796, WO94/23697, WO91/14445 and EP-524,968. As described in U.S. S No. 60/023,867, on non-viral delivery, the nucleic acid sequences encoding a polypeptide can be inserted into conventional vectors that contain conventional control sequences for high level expression, and then be incubated with synthetic gene transfer molecules such as polymeric DNA-binding cations like polylysine, protamine, and albumin, linked to cell targeting ligands such as asialoorosomucoid, insulin, galactose, lactose, or transferrin. Other delivery systems include the use of liposomes to encapsulate DNA comprising the gene under the control of a variety of tissue-specific or ubiquitously-active promoters. Further non-viral delivery suitable for use includes mechanical delivery systems such as the approach described in Woffendin et al (1994) Proc. Natl. Acad. Sci. USA 91(24):11581-11585. Moreover, the coding sequence and the product of expression of such can be delivered through deposition of photopolymerized hydrogel materials. Other conventional methods for gene delivery that can be used for delivery of the coding sequence include, for example, use of hand-held gene transfer particle gun, as described in U.S. Pat. No. 5,149,655; use of ionizing radiation for activating transferred gene, as described in U.S. Pat. No. 5,206,152 and WO92/11033

Exemplary liposome and polycationic gene delivery vehicles are those described in U.S. Pat. Nos. 5,422,120 and 4,762,915; in WO 95/13796; WO94/23697, and WO91/14445; in EP-0524968; and in Stryer, Biochemistry, pages 236-240 (1975) W.H. Freeman, San Francisco; Szoka (1980) Biochem Biophys Acta 600:1; Bayer (1979) Biochem Biophys Acta 550:464; Rivnay (1987) Meth Enzymol 149:119; Wang (1987) Proc Natl Acad Sci 84:7851; Plant (1989) Anal Biochem 176:420.

A polynucleotide composition can comprises therapeutically effective amount of a gene therapy vehicle, as the term is defined above. For purposes of the present invention, an effective dose will be from about 0.01 mg/kg to 50 mg/kg or 0.05 mg/kg to about 10 mg/kg of the DNA constructs in the individual to which it is administered.

Delivery Methods

Once formulated, the polynucleotide compositions of the invention can be administered (1) directly to the subject; (2) delivered ex vivo, to cells derived from the subject; or (3) in vitro for expression of recombinant proteins. The subjects to be treated can be mammals or birds. Also, human subjects can be treated.

Direct delivery of the compositions will generally be accomplished by injection, either subcutaneously, intraperitoneally, intravenously or intramuscularly or delivered to the interstitial space of a tissue. The compositions can also be administered into a lesion. Other modes of administration include oral and pulmonary administration, suppositories, and transdermal or transcutaneous applications (eg. see WO98/20734), needles, and gene guns or hyposprays. Dosage treatment may be a single dose schedule or a multiple dose schedule.

Methods for the ex vivo delivery and reimplantation of transformed cells into a subject are known in the art and described in eg. WO93/14778. Examples of cells useful in ex vivo applications include, for example, stem cells, particularly hematopoetic, lymph cells, macrophages, dendritic cells, or tumor cells.

Generally, delivery of nucleic acids for both ex vivo and in vitro applications can be accomplished by the following procedures, for example, dextran-mediated transfection, calcium phosphate precipitation, polybrene mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide(s) in liposomes, and direct microinjection of the DNA into nuclei, all well known in the art.

Polynucleotide and Polypeptide Pharmaceutical Compositions

In addition to the pharmaceutically acceptable carriers and salts described above, the following additional agents can be used with polynucleotide and/or polypeptide compositions.

A. Polypeptides

One example are polypeptides which include, without limitation: asioloorosomucoid (ASOR); transferrin; asialoglycoproteins; antibodies; antibody fragments; ferritin; interleukins; interferons, granulocyte, macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), macrophage colony stimulating factor (M-CSF), stem cell factor and erythropoietin. Viral antigens, such as envelope proteins, can also be used. Also, proteins from other invasive organisms, such as the 17 amino acid peptide from the circumsporozoite protein of plasmodium falciparum known as RII.

B. Hormones, Vitamins, etc.

Other groups that can be included are, for example: hormones, steroids, androgens, estrogens, thyroid hormone, or vitamins, folic acid.

C. Polyalkylenes, Polysaccharides, etc.

Also, polyalkylene glycol can be included with the desired polynucleotides/polypeptides. In a preferred embodiment, the polyalkylene glycol is polyethlylene glycol. In addition, mono-, di-, or polysaccharides can be included. In a preferred embodiment of this aspect, the polysaccharide is dextran or DEAE-dextran. Also, chitosan and poly(lactide-co-glycolide)

D. Lipids, and Liposomes

The desired polynucleotide/polypeptide can also be encapsulated in lipids or packaged in liposomes prior to delivery to the subject or to cells derived therefrom.

Lipid encapsulation is generally accomplished using liposomes which are able to stably bind or entrap and retain nucleic acid. The ratio of condensed polynucleotide to lipid preparation can vary but will generally be around 1:1 (mg DNA:micromoles lipid), or more of lipid. For a review of the use of liposomes as carriers for delivery of nucleic acids, see, Hug and Sleight (1991) Biochim. Biophys. Acta. 1097:1-17; Straubinger (1983) Meth. Enzymol. 101:512-527.

Liposomal preparations for use in the present invention include cationic (positively charged), anionic (negatively charged) and neutral preparations. Cationic liposomes have been shown to mediate intracellular delivery of plasmid DNA (Felgner (1987) Proc. Natl. Acad. Sci. USA 84:7413-7416); mRNA (Malone (1989) Proc. Natl. Acad. Sci. USA 86:6077-6081); and purified transcription factors (Debs (1990) J. Biol. Chem. 265:10189-10192), in functional form.

Cationic liposomes are readily available. For example, N[1-2,3-dioleyloxy)propyl]-N,N,N-triethylammonium (DOTMA) liposomes are available under the trademark Lipofectin, from GIBCO BRL, Grand Island, N.Y. (See, also, Felgner supra). Other commercially available liposomes include transfectace (DDAB/DOPE) and DOTAP/DOPE (Boerhinger). Other cationic liposomes can be prepared from readily available materials using techniques well known in the art. See, eg. Szoka (1978) Proc. Nail. Acad. Sci. USA 75:41944198; WO90/11092 for a description of the synthesis of DOTAP (1,2-bis(oleoyloxy)-3-(trimethylammonio)propane) liposomes.

Similarly, anionic and neutral liposomes are readily available, such as from Avanti Polar Lipids (Birmingham, Ala.), or can be easily prepared using readily available materials. Such materials include phosphatidyl choline, cholesterol, phosphatidyl ethanolamine, dioleoylphosphatidyl choline (DOPC), dioleoylphosphatidyl glycerol (DOPG), dioleoylphoshatidyl ethanolamine (DOPE), among others. These materials can also be mixed with the DOTMA and DOTAP starting materials in appropriate ratios. Methods for making liposomes using these materials are well known in the art.

The liposomes can comprise multilammelar vesicles (MLVs), small unilamellar vesicles (SUVs), or large unilamellar vesicles (LUVs). The various liposome-nucleic acid complexes are prepared using methods known in the art See eg. Straubinger (1983) Meth. Immunol. 101:512-527; Szoka (1978) Proc. Natl. Acad. Sci. USA 75:4194-4198; Papahadjopoulos (1975) Biochim. Biophys. Acta 394:483; Wilson (1979) Cell 17:77); Deamer & Bangham (1976) Biochim. Biophys. Acia 443:629; Ostro (1977) Biochem. Biophys. Res. Commun. 76:836; Fraley (1979) Proc. Natl. Acad. Sci. USA 76:3348); Enoch & Strittmatter (1979) Proc. Natl. Acad. Sci. USA 76:145; Fraley (1980) J. Biol. Chem. (1980) 255:10431; Szoka & Papahadjopoulos (1978) Proc. Natl. Acad. Sci. USA 75:145; and Schaefer-Ridder (1982) Science 215:166.

E. Lipoproteins

In addition, lipoproteins can be included with the polynucleotide/polypeptide to be delivered. Examples of lipoproteins to be utilized include: chylomicrons, HDL, DL, LDL, and VLDL. Mutants, fragments, or fusions of these proteins can also be used. Also, modifications of naturally occurring lipoproteins can be used, such as acetylated LDL. These lipoproteins can target the delivery of polynucleotides to cells expressing lipoprotein receptors. Preferably, if lipoproteins are including with the polynucleotide to be delivered, no other targeting ligand is included in the composition.

Naturally occurring lipoproteins comprise a lipid and a protein portion. The protein portion are known as apoproteins. At the present, apoproteins A, B, C, D, and E have been isolated and identified. At least two of these contain several proteins, designated by Roman numerals, AI, AII, AIV; CI, CII, CIII.

A lipoprotein can comprise more than one apoprotein. For example, naturally occurring chylomicrons comprises of A, B, C, and E, over time these lipoproteins lose A and acquire C and E apoproteins. VLDL comprises A, B, C, and E apoproteins, LDL comprises apoprotein B; and HDL comprises apoproteins A, C, and E.

The amino acid of these apoproteins are known and are described in, for example, Breslow (1985) Annu Rev. Biochem 54:699; Law (1986) Adv. Exp Med. Biol. 151:162; Chen (1986) J Biol Chem 261:12918; Kane (1980) Proc Natl Acad Sci USA 77:2465; and Utermann (1984) Hum Genet 65:232.

Lipoproteins contain a variety of lipids including, triglycerides, cholesterol (free and esters), and phospholipids. The composition of the lipids varies in naturally occurring lipoproteins. For example, chylomicrons comprise mainly triglycerides. A more detailed description of the lipid content of naturally occurring lipoproteins can be found, for example, in Meth. Enzymol. 128 (1986). The composition of the lipids are chosen to aid in conformation of the apoprotein for receptor binding activity. The composition of lipids can also be chosen to facilitate hydrophobic interaction and association with the polynucleotide binding molecule.

Naturally occurring lipoproteins can be isolated from serum by ultracentrifugation, for instance. Such methods are described in Meth. Enzymol. (supra); Pitas (1980) J. Biochem. 255:5454-5460 and Mahey (1979) J. Clin. Invest 64:743-750. Lipoproteins can also be produced by in vitro or recombinant methods by expression of the apoprotein genes in a desired host cell. See, for example, Atkinson (1986) Annu Rev Biophys Chem 15:403 and Radding (1958) Biochim Biophys Acia 30: 443. Lipoproteins can also be purchased from commercial suppliers, such as Biomedical Techniologies, Inc., Stoughton, Mass., USA. Further description of lipoproteins can be found in Zuckermann et al. PCT/US97/14465.

F. Polycationic Agents

Polycationic agents can be included, with or without lipoprotein, in a composition with the desired polynucleotide/polypeptide to be delivered.

Polycationic agents, typically, exhibit a net positive charge at physiological relevant pH and are capable of neutralizing the electrical charge of nucleic acids to facilitate delivery to a desired location. These agents have both in vitro, ex vivo, and in vivo applications. Polycationic agents can be used to deliver nucleic acids to a living subject either intramuscularly, subcutaneously, etc.

The following are examples of useful polypeptides as polycationic agents: polylysine, polyarginine, polyornithine, and protamine. Other examples include histones, protamines, human serum albumin, DNA binding proteins, non-histone chromosomal proteins, coat proteins from DNA viruses, such as (X174, transcriptional factors also contain domains that bind DNA and therefore may be useful as nucleic aid condensing agents. Briefly, transcriptional factors such as C/CEBP, c-jun, c-fos, AP-1, AP-2, AP-3, CPF, Prot-1, Sp-1, Oct-1, Oct-2, CREP, and TFIID contain basic domains that bind DNA sequences.

Organic polycationic agents include: spermine, spermidine, and purtrescine.

The dimensions and of the physical properties of a polycationic agent can be extrapolated from the list above, to construct other polypeptide polycationic agents or to produce synthetic polycationic agents.

Synthetic polycationic agents which are useful include, for example, DEAE-dextran, polybrene. Lipofectin™, and lipofectAMINE™ are monomers that form polycationic complexes when combined with polynucleotides/polypeptides.

Immunodiagnostic Assays

Neisserial antigens of the invention can be used in immunoassays to detect antibody levels (or, conversely, anti-Neisserial antibodies can be used to detect antigen levels). Immunoassays based on well defined, recombinant antigens can be developed to replace invasive diagnostics methods. Antibodies to Neisserial proteins within biological samples, including for example, blood or serum samples, can be detected. Design of the immunoassays is subject to a great deal of variation, and a variety of these are known in the art. Protocols for the immunoassay may be based, for example, upon competition, or direct reaction, or sandwich type assays. Protocols may also, for example, use solid supports, or may be by immunoprecipitation. Most assays involve the use of labeled antibody or polypeptide; the labels may be, for example, fluorescent, chemiluminescent, radioactive, or dye molecules. Assays which amplify the signals from the probe are also known; examples of which are assays which utilize biotin and avidin, and enzyme-labeled and mediated immunoassays, such as ELISA assays.

Kits suitable for immunodiagnosis and containing the appropriate labeled reagents are constructed by packaging the appropriate materials, including the compositions of the invention, in suitable containers, along with the remaining reagents and materials (for example, suitable buffers, salt solutions, etc.) required for the conduct of the assay, as well as suitable set of assay instructions.

Nucleic Acid Hybridisation

“Hybridization” refers to the association of two nucleic acid sequences to one another by hydrogen bonding. Typically, one sequence will be fixed to a solid support and the other will be free in solution. Then, the two sequences will be placed in contact with one another under conditions that favor hydrogen bonding. Factors that affect this bonding include: the type and volume of solvent; reaction temperature; time of hybridization; agitation; agents to block the non-specific attachment of the liquid phase sequence to the solid support (Denhardt's reagent or BLOTTO); concentration of the sequences; use of compounds to increase the rate of association of sequences (dextran sulfate or polyethylene glycol); and the stringency of the washing conditions following hybridization. See Sambrook et al. [supra] Volume 2, chapter 9, pages 9.47 to 9.57.

“Stringency” refers to conditions in a hybridization reaction that favor association of very similar sequences over sequences that differ. For example, the combination of temperature and salt concentration should be chosen that is approximately 120 to 200° C. below the calculated Tm of the hybrid under study. The temperature and salt conditions can often be determined empirically in preliminary experiments in which samples of genomic DNA immobilized on filters are hybridized to the sequence of interest and then washed under conditions of different stringencies. See Sambrook et al. at page 9.50.

Variables to consider when performing, for example, a Southern blot are (1) the complexity of the DNA being blotted and (2) the homology between the probe and the sequences being detected. The total amount of the fragment(s) to be studied can vary a magnitude of 10, from 0.1 to 1 g for a plasmid or phage digest to 10⁻⁹ to 10⁻⁸ g for a single copy gene in a highly complex eukaryotic genome. For lower complexity polynucleotides, substantially shorter blotting, hybridization, and exposure times, a smaller amount of starting polynucleotides, and lower specific activity of probes can be used. For example, a single-copy yeast gene can be detected with an exposure time of only 1 hour starting with 1 μg of yeast DNA, blotting for two hours, and hybridizing for 4-8 hours with a probe of 10⁸ cpm/μg. For a single-copy mammalian gene a conservative approach would start with 10 μg of DNA, blot overnight, and hybridize overnight in the presence of 10% dextran sulfate using a probe of greater than 10⁸ cpm/μg, resulting in an exposure time of 24 hours.

Several factors can affect the melting temperature (Tm) of a DNA-DNA hybrid between the probe and the fragment of interest, and consequently, the appropriate conditions for hybridization and washing. In many cases the probe is not 100% homologous to the fragment. Other commonly encountered variables include the length and total G+C content of the hybridizing sequences and the ionic strength and formamide content of the hybridization buffer. The effects of all of these factors can be approximated by a single equation: Tm—81+16.6(log₁₀ Ci)+0.4[% (G+C)]−0.6(% formamide)-600/n−1.5(% mismatch). where Ci is the salt concentration (monovalent ions) and n is the length of the hybrid in base pairs (slightly modified from Meinkoth & Wahl (1984) Anal. Biochem. 138: 267-284).

In designing a hybridization experiment, some factors affecting nucleic acid hybridization can be conveniently altered. The temperature of the hybridization and washes and the salt concentration during the washes are the simplest to adjust. As the temperature of the hybridization increases (ie. stringency), it becomes less likely for hybridization to occur between strands that are nonhomologous, and as a result, background decreases. If the radiolabeled probe is not completely homologous with the immobilized fragment (as is frequently the case in gene family and interspecies hybridization experiments), the hybridization temperature must be reduced, and background will increase. The temperature of the washes affects the intensity of the hybridizing band and the degree of background in a similar manner. The stringency of the washes is also increased with decreasing salt concentrations.

In general, convenient hybridization temperatures in the presence of 50% formamide are 42° C. for a probe with is 95% to 100% homologous to the target fragment, 37° C. for 90% to 95% homology, and 32° C. for 85% to 90% homology. For lower homologies, formamide content should be lowered and temperature adjusted accordingly, using the equation above. If the homology between the probe and the target fragment are not known, the simplest approach is to start with both hybridization and wash conditions which are nonstringent. If non-specific bands or high background are observed after autoradiography, the filter can be washed at high stringency and reexposed. If the time required for exposure makes this approach impractical, several hybridization and/or washing stringencies should be tested in parallel.

Nucleic Acid Probe Assays

Methods such as PCR, branched DNA probe assays, or blotting techniques utilizing nucleic acid probes according to the invention can determine the presence of cDNA or mRNA. A probe is said to “hybridize” with a sequence of the invention if it can form a duplex or double stranded complex, which is stable enough to be detected.

The nucleic acid probes will hybridize to the Neisserial nucleotide sequences of the invention (including both sense and antisense strands). Though many different nucleotide sequences will encode the amino acid sequence, the native Neisserial sequence is preferred because it is the actual sequence present in cells. mRNA represents a coding sequence and so a probe should be complementary to the coding sequence; single-stranded cDNA is complementary to mRNA, and so a cDNA probe should be complementary to the non-coding sequence.

The probe sequence need not be identical to the Neisserial sequence (or its complement)—some variation in the sequence and length can lead to increased assay sensitivity if the nucleic acid probe can form a duplex with target nucleotides, which can be detected. Also, the nucleic acid probe can include additional nucleotides to stabilize the formed duplex. Additional Neisserial sequence may also be helpful as a label to detect the formed duplex. For example, a non-complementary nucleotide sequence may be attached to the 5′ end of the probe, with the remainder of the probe sequence being complementary to a Neisserial sequence. Alternatively, non-complementary bases or longer sequences can be interspersed into the probe, provided that the probe sequence has sufficient complementarity with the a Neisserial sequence in order to hybridize therewith and thereby form a duplex which can be detected.

The exact length and sequence of the probe will depend on the hybridization conditions, such as temperature, salt condition and the like. For example, for diagnostic applications, depending on the complexity of the analyte sequence, the nucleic acid probe typically contains at least 10-20 nucleotides, preferably 15-25, and more preferably at least 30 nucleotides, although it may be shorter than this. Short primers generally require cooler temperatures to form sufficiently stable hybrid complexes with the template.

Probes may be produced by synthetic procedures, such as the triester method of Matteucci et al. [J. Am. Chem. Soc. (1981) 103:3185], or according to Urdea et al. [Proc. Natl. Acad. Sci. USA (1983) 80: 7461], or using commercially available automated oligonucleotide synthesizers.

The chemical nature of the probe can be selected according to preference. For certain applications, DNA or RNA are appropriate. For other applications, modifications may be incorporated eg. backbone modifications, such as phosphorothioates or methylphosphonates, can be used to increase in vivo half-life, alter RNA affinity, increase nuclease resistance etc. [eg. see Agrawal & Iyer (1995) Curr Opin Biotechnol 6:12-19; Agrawal (1996) TIBTECH 14:376-387]; analogues such as peptide nucleic acids may also be used [eg. see Corey (1997) TIBTECH 15:224-229; Buchardt et al. (1993) TIBTECH 11:384-386].

Alternatively, the polymerase chain reaction (PCR) is another well-known means for detecting small amounts of target nucleic acids. The assay is described in: Mullis et al. [Meth. Enzymol. (1987) 155: 335-350]; U.S. Pat. Nos. 4,683,195 and 4,683,202. Two “primer” nucleotides hybridize with the target nucleic acids and are used to prime the reaction. The primers can comprise sequence that does not hybridize to the sequence of the amplification target (or its complement) to aid with duplex stability or, for example, to incorporate a convenient restriction site. Typically, such sequence will flank the desired Neisserial sequence.

A thermostable polymerase creases copies of target nucleic acids from the primers using the original target nucleic acids as a template. After a threshold amount of target nucleic acids are generated by the polymerase, they can be detected by more traditional methods, such as Southern blots. When using the Southern blot method, the labelled probe will hybridize to the Neisserial sequence (or its complement).

Also, mRNA or cDNA can be detected by traditional blotting techniques described in Sambrook et al [supra]. mRNA, or cDNA generated from mRNA using a polymerase enzyme, can be purified and separated using gel electrophoresis. The nucleic acids on the gel are then blotted onto a solid support, such as nitrocellulose. The solid support is exposed to a labelled probe and then washed to remove any unhybridized probe. Next, the duplexes containing the labeled probe are detected. Typically, the probe is labelled with a radioactive moiety.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1-7 show biochemical data and sequence analysis pertaining to Examples 1, 2, 3, 7, 13, 16 and 19, respectively, with ORFs 40, 38, 44, 52, 114, 41 and 124. M1 and M2 are molecular weight markers. Arrows indicate the position of the main recombinant product or, in Western blots, the position of the main N. meningitidis immunoreactive band. TP indicates N. meningitidis total protein extract; OMV indicates N. meningitidis outer membrane vesicle preparation. In bactericidal assay results: a diamond (♦) shows preimmune data; a triangle (▴) shows GST control data; a circle (●) shows data with recombinant N. meningitidis protein. Computer analyses show a hydrophilicity plot (upper), an antigenic index plot (middle), and an AMPHI analysis (lower). The AMPHI program has been used to predict T-cell epitopes [Gao et al. (1989) J. Immunol. 143:3007; Roberts et al. (1996) AIDS Res Hum Retrovir 12:593; Quakyi et al. (1992) Scand J Immunol suppl. 11:9) and is available in the Protean package of DNASTAR, Inc. (1228 South Park Street, Madison, Wis. 53715 USA).

FIG. 8 shows an alignment comparison of amino acid sequences for ORF 40 for several strains of Neisseria. Dark shading indicates regions of homology, and gray shading indicates the conservation of amino acids with similar characteristics. The Figure demonstrates a high degree of conservation among the various strains, further confirming its utility as an antigen for both vaccines and diagnostics.

EXAMPLES

The examples describe nucleic acid sequences which have been identified in N. meningitidis, along with their putative translation products. Not all of the nucleic acid sequences are complete ie. they encode less than the full-length wild-type protein. It is believed at present that none of the DNA sequences described herein have significant homologs in N. gonorrhoeae.

The examples are generally in the following format:

-   -   a nucleotide sequence which has been identified in N.         meningitidis (strain B)     -   the putative translation product of this sequence     -   a computer analysis of the translation product based on database         comparisons     -   a corresponding gene and protein sequence identified in N.         meningitidis (strain A)     -   a description of the characteristics of the proteins which         indicates that they might be suitably antigenic     -   results of biochemical analysis (expression, purification,         ELISA, FACS etc.)

The examples typically include details of sequence homology between species and strains. Proteins that are similar in sequence are generally similar in both structure and function, and the homology often indicates a common evolutionary origin. Comparison with sequences of proteins of known function is widely used as a guide for the assignment of putative protein function to a new sequence and has proved particularly useful in whole-genome analyses.

Sequence comparisons were performed at NCBI (http://www.ncbi.nlm.nih.gov) using the algorithms BLAST, BLAST2, BLASTn, BLASTp, tBLASTn, BLASTx, & tBLASTx [eg. see also Altschul et al. (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Research 25:2289-3402]. Searches were performed against the following databases: non-redundant GenBank+EMBL+DDBJ+PDB sequences and non-redundant GenBank CDS translations+PDB+SwissProt+SPupdate+PIR sequences.

Dots within nucleotide sequences (eg. position 288 in Example 12) represent nucleotides which have been arbitrarily introduced in order to maintain a reading frame. In the same way, double-underlined nucleotides were removed. Lower case letters (eg. position 589 in Example 12) represent ambiguities which arose during alignment of independent sequencing reactions (some of the nucleotide sequences in the examples are derived from combining the results of two or more experiments).

Nucleotide sequences were scanned in all six reading frames to predict the presence of hydrophobic domains using an algorithm based on the statistical studies of Esposti et al. [Critical evaluation of the hydropathy of membrane proteins (1990) Eur J Biochem 190:207-219]. These domains represent potential transmembrane regions or hydrophobic leader sequences.

Open reading frames were predicted from fragmented nucleotide sequences using the program ORFFINDER (NCBI).

Underlined amino acid sequences indicate possible transmembrane domains or leader sequences in the ORFs, as predicted by the PSORT algorithm (http://www.psort.nibb.ac.jp). Functional domains were also predicted using the MOTIFS program (GCG Wisconsin & PROSITE).

Various tests can be used to assess the in vivo immunogenicity of the proteins identified in the examples. For example, the proteins can be expressed recombinantly and used to screen patient sera by immunoblot A positive reaction between the protein and patient serum indicates that the patient has previously mounted an immune response to the protein in question ie. the protein is an immunogen. This method can also be used to identify immunodominant proteins.

The recombinant protein can also be conveniently used to prepare antibodies eg. in a mouse. These can be used for direct confirmation that a protein is located on the cell-surface. Labelled antibody (eg. fluorescent labelling for FACS) can be incubated with intact bacteria and the presence of label on the bacterial surface confirms the location of the protein.

In particular, the following methods (A) to (S) were used to express, purify and biochemically characterise the proteins of the invention:

A) Chromosomal DNA Preparation

N. meningitidis strain 2996 was grown to exponential phase in 100 ml of GC medium, harvested by centrifugation, and resuspended in 5 ml buffer (20% Sucrose, 50 mM Tris-HCl, 50 mM EDTA, pH8). After 10 minutes incubation on ice, the bacteria were lysed by adding 10 ml lysis solution (50 mM NaCl, 1% Na-Sarkosyl, 50 μg/ml Proteinase K), and the suspension was incubated at 37° C. for 2 hours. Two phenol extractions (equilibrated to pH 8) and one ChCl₃/isoamylalcohol (24:1) extraction were performed. DNA was precipitated by addition of 0.3M sodium acetate and 2 volumes ethanol, and was collected by centrifugation. The pellet was washed once with 70% ethanol and redissolved in 4 ml buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8). The DNA concentration was measured by reading the OD at 260 nm.

B) Oligonucleotide Design

Synthetic oligonucleotide primers were designed on the basis of the coding sequence of each ORF, using (a) the meningococcus B sequence when available, or (b) the gonococcus/meningococcus A sequence, adapted to the codon preference usage of meningococcus as necessary. Any predicted signal peptides were omitted, by deducing the 5′-end amplification primer sequence immediately downstream from the predicted leader sequence.

The 5′ primers included two restriction enzyme recognition sites (BamHI-NdeI, BamHI-NheI, or EcoRI-NheI, depending on the gene's own restriction pattern); the 3′ primers included a XhoI restriction site. This procedure was established in order to direct the cloning of each amplification product (corresponding to each ORF) into two different expression systems: pGEX-KG (using either BamHI-XhoI or EcoRI-XhoI), and pET21b+ (using either NdeI-XhoI or NheI-XhoI). 5′end primer tail: CGCGGATCCCATATG (BamHI-NdeI) CGCGGATCCGCTAGC (BamHI-NheI) CCGGAATTCTAGCTAGC (EcoRI-NheI) 3′-end primer tail: CCCGCTCGAG (XhoI)

As well as containing the restriction enzyme recognition sequences, the primers included nucleotides which hybridised to the sequence to be amplified. The number of hybridizing nucleotides depended on the melting temperature of the whole primer, and was determined for each primer using the formulae: T _(m)=4 (G+C)+2 (A+T)  (tail excluded) T _(m)=64.9+0.41 (% GC)−600/N  (whole primer)

The average melting temperature of the selected oligos were 65-70° C. for the whole oligo and 50-55° C. for the hybridising region alone.

Table I shows the forward and reverse primers used for each amplification. Oligos were synthesized by a Perkin Elmer 394 DNA/RNA Synthesizer, eluted from the columns in 2 ml NH₄OH, and deprotected by 5 hours incubation at 56° C. The oligos were precipitated by addition of 0.3M Na-Acetate and 2 volumes ethanol. The samples were then centrifuged and the pellets resuspended in either 100 μl or 1 ml of water. OD₂₆₀ was determined using a Perkin Elmer Lambda Bio spectrophotometer and the concentration was determined and adjusted to 2-10 pmol/μl. TABLE I PCR primers ORF Primer Sequence Restriction sites ORF 38 Forward CGCGGATCCCATATG-TCGCCGCAAAATTCCGA BamHI-NdeI <SEQ ID 112> Reverse CCCGCTCGAG-TTTTGCCGCGTTAAAAGC XhoI <SEQ ID 113> ORF 40 Forward CGCGGATCCCATATG-ACCGTGAAGACCGCC BamHI-NdeI <SEQ ID 114> Reverse CCCGCTCGAG-CCACTGATAACCGACAGA XhoI <SEQ ID 115> ORF 41 Forward CGCGGATCCCATATG-TATTTGAAACAGCTCCAAG BamHI-NdeI <SEQ ID 116> Reverse CCCGCTCGAG-TTCTGGGTGAATGTTA XhoI <SEQ ID 117> ORF 44 Forward GCGGATCCCATATG-GGCACGGACAACCCC BamHI-NdeI <SEQ ID 118> Reverse CCCGCTCGAG-ACGTGGGGAACAGTCT XhoI <SEQ ID 119> ORF 51 Forward GCGGATCCCATATG-AAAAATATTCAAGTAGTTGC BamHI-NdeI <SEQ ID 120> Reverse CCCGCTCGAG-AAGTTTGATTAAACCCG XhoI <SEQ ID 121> ORF 52 Forward CGCGGATCCCATATG-TGCCAACCGCAATCCG BamHI-NdeI <SEQ ID 122> Reverse CCCGCTCGAG-TTTTTCCAGCTCCGGCA XhoI <SEQ ID 123> ORF 56 Forward GCGGATCCCATATG-GTTATCGGAATATTACTCG BamHI-NdeI <SEQ ID 124> Reverse CCCGCTCGAG-GGCTGCAGAAGCTGG XhoI <SEQ ID 125> ORF 69 Forward CGCGGATCCCATATG-CGGACGTGGTTGGTTTT BamHI-NdeI <SEQ ID 126> Reverse CCCGCTCGAG-ATATCTTCCGTTTTTTTCAC XhoI <SEQ ID 127> ORF 82 Forward CGCGGATCCGCTAGC-GTAAATTTATTATTTTTAGAA BamHI-NheI <SEQ ID 128> Reverse CCCGCTCGAG-TCCAACTCATTGAAGTA XhoI <SEQ ID 129> ORF 114 Forward CGCGGATCCCATATG-AATAAAGGTTTACATCGCAT BamHI-NheI <SEQ ID 130> Reverse CCCGCTCGAG-AATCGCTGCACCGGCT XhoI <SEQ ID 131> ORF 124 Forward CGCGGATCCCATATG-ACTGCCTTTTCGACA BamHI-NheI <SEQ ID 132> Reverse CCCGCTCGAG-GCGTGAAGCGTCAGGA XhoI <SEQ ID 133> C) Amplification

The standard PCR protocol was as follows: 50-200 ng of genomic DNA were used as a template in the presence of 20-40 μM of each oligo, 400-8004M dNTs solution, 1×PCR buffer (including 1.5 mM MgCl₂), 2.5 units TaqI DNA polymerase (using Perkin-Elmer AmpliTaQ, GIBCO Platinum, Pwo DNA polymerase, or Tahara Shuzo Taq polymerase).

In some cases, PCR was optimised by the addition of 10 μl DMSO or 50 μl 2M betaine.

After a hot start (adding the polymerase during a preliminary 3 minute incubation of the whole mix at 95° C.), each sample underwent a double-step amplification: the first 5 cycles were performed using as the hybridization temperature the one of the oligos excluding the restriction enzymes tail, followed by 30 cycles performed according to the hybridization temperature of the whole length oligos. The cycles were followed by a final 10 minute extension step at 72° C.

The standard cycles were as follows: Denaturation Hybridisation Elongation First 5 cycles 30 seconds 30 seconds 30-60 seconds 95° C. 50-55° C. 72° C. Last 30 cycles 30 seconds 30 seconds 30-60 seconds 95° C. 65-70° C. 72° C.

The elongation time varied according to the length of the ORF to be amplified.

The amplifications were performed using either a 9600 or a 2400 Perkin Elmer GeneAmp PCR System. To check the results, 1/10 of the amplification volume was loaded onto a 1-1.5% agarose gel and the size of each amplified fragment compared with a DNA molecular weight marker.

The amplified DNA was either loaded directly on a 1% agarose gel or first precipitated with ethanol and resuspended in a suitable volume to be loaded on a 1% agarose gel. The DNA fragment corresponding to the right size band was then eluted and purified from gel, using the Qiagen Gel Extraction Kit, following the instructions of the manufacturer. The final volume of the DNA fragment was 30 μl or 500 of either water or 10 mM Tris, pH 8.5.

D) Digestion of PCR Fragments

The purified DNA corresponding to the amplified fragment was split into 2 aliquots and double-digested with:

-   -   NdeI/XhoI or NheI/XhoI for cloning into pET-21b+ and further         expression of the protein as a C-terminus His-tag fusion     -   BamHI/XhoI or EcoRI/XhoI for cloning into pGEX-KG and further         expression of the protein as N-terminus GST fusion.     -   EcoRI/PstI, EcoRI/SalI, SalI/PstI for cloning into pGex-His and         further expression of the protein as N-terminus His-tag fusion

Each purified DNA fragment was incubated (37° C. for 3 hours to overnight) with 20 units of each restriction enzyme (New England Biolabs) in a either 30 or 40 μl final volume in the presence of the appropriate buffer. The digestion product was then purified using the QIAquick PCR purification kit, following the manufacturer's instructions, and eluted in a final volume of 30 or 50 μl of either water or 10 mM Tris-HCl, pH 8.5. The final DNA concentration was determined by 1% agarose gel electrophoresis in the presence of titrated molecular weight marker.

E) Digestion of the Cloning Vectors (pET22B, pGEX-KG, pTRC-His A, and pGex-His)

10 μg plasmid was double-digested with 50 units of each restriction enzyme in 200 μl reaction volume in the presence of appropriate buffer by overnight incubation at 37° C. After loading the whole digestion on a 1% agarose gel, the band corresponding to the digested vector was purified from the gel using the Qiagen QIAquick-Gel Extraction Kit and the DNA was eluted in 50 μl of 10 mM Tris-HCl, pH 8.5. The DNA concentration was evaluated by measuring OD₂₆₀ of the sample, and adjusted to 50 μg/μl. 1 μl of plasmid was used for each cloning procedure.

The vector pGEX-His is a modified pGEX-2T vector carrying a region encoding six histidine residues upstream to the thrombin cleavage site and containing the multiple cloning site of the vector pTRC99 (Pharmacia).

F) Cloning

The fragments corresponding to each ORF, previously digested and purified, were ligated in both pET22b and pGEX-KG. In a final volume of 20 μl, a molar ratio of 3:1 fragment/vector was ligated using 0.5 μl of NEB T4 DNA ligase (400 units/μl), in the presence of the buffer supplied by the manufacturer. The reaction was incubated at room temperature for 3 hours. In some experiments, ligation was performed using the Boehringer “Rapid Ligation Kit”, following the manufacturer's instructions.

In order to introduce the recombinant plasmid in a suitable strain, 100 μl E. coli DH5 competent cells were incubated with the ligase reaction solution for 40 minutes on ice, then at 37° C. for 3 minutes, then, after adding 800 μl LB broth, again at 37° C. for 20 minutes. The cells were then centrifuged at maximum speed in an Eppendorf microfuge and resuspended in approximately 200 μl of the supernatant. The suspension was then plated on LB ampicillin (100 mg/ml).

The screening of the recombinant clones was performed by growing 5 randomly-chosen colonies overnight at 37° C. in either 2 ml (pGEX or pTC clones) or 5 ml (pET clones) LB broth+100 μg/ml ampicillin. The cells were then pelletted and the DNA extracted using the Qiagen QIAprep Spin Miniprep Kit, following the manufacturer's instructions, to a final volume of 30 μl. 5 μl of each individual miniprep (approximately 1 g) were digested with either NdeI/XhoI or BamHI/XhoI and the whole digestion loaded onto a 1-1.5% agarose gel (depending on the expected insert size), in parallel with the molecular weight marker (1 Kb DNA Ladder, GIBCO). The screening of the positive clones was made on the base of the correct insert size.

G) Expression

Each ORF cloned into the expression vector was transformed into the strain suitable for expression of the recombinant protein product. 1 μl of each construct was used to transform 30 μl of E. coli BL21 (pGEX vector), E. coli TOP 10 (pTRC vector) or E. coli BL21-DE3 (pET vector), as described above. In the case of the pGEX-His vector, the same E. coli strain (W3110) was used for initial cloning and expression. Single recombinant colonies were inoculated into 2 ml LB+Amp (100 μg/ml), incubated at 37° C. overnight, then diluted 1:30 in 20 ml of LB+Amp (100 μg/ml) in 100 ml flasks, making sure that the OD₆₀₀ ranged between 0.1 and 0.15. The flasks were incubated at 30° C. into gyratory water bath shakers until OD indicated exponential growth suitable for induction of expression (0.4-0.8 OD for pET and pTRC vectors; 0.8-1 OD for pGEX and pGEX-His vectors). For the pET, pTRC and pGEX-His vectors, the protein expression was induced by addition of 1 mM IPTG, whereas in the case of pGEX system the final concentration of IPTG was 0.2 mM. After 3 hours incubation at 30° C., the final concentration of the sample was checked by OD. In order to check expression, 1 ml of each sample was removed, centrifuged in a microfuge, the pellet resuspended in PBS, and analysed by 12% SDS-PAGE with Coomassie Blue staining. The whole sample was centrifuged at 6000 g and the pellet resuspended in PBS for further use.

H) GST-Fusion Proteins Large-Scale Purification.

A single colony was grown overnight at 37° C. on LB+Amp agar plate. The bacteria were inoculated into 20 ml of LB+Amp liquid culture in a water bath shaker and grown overnight. Bacteria were diluted 1:30 into 600 ml of fresh medium and allowed to grow at the optimal temperature (20-37° C.) to OD₅₅₀ 0.8-1. Protein expression was induced with 0.2 mM IPTG followed by three hours incubation. The culture was centrifuged at 800 rpm at 4° C. The supernatant was discarded and the bacterial pellet was resuspended in 7.5 ml cold PBS. The cells were disrupted by sonication on ice for 30 sec at 40 W using a Branson sonifier B-15, frozen and thawed twice and centrifuged again. The supernatant was collected and mixed with 150 μl Glutatione-Sepharose 4B resin (Pharmacia) (previously washed with PBS) and incubated at room temperature for 30 minutes. The sample was centrifuged at 700 g for 5 minutes at 4° C. The resin was washed twice with 10 ml cold PBS for 10 minutes, resuspended in 1 ml cold PBS, and loaded on a disposable column. The resin was washed twice with 2 ml cold PBS until the flow-through reached OD₂₈₀ of 0.02-0.06. The GST-fusion protein was eluted by addition of 70011 cold Glutathione elution buffer (10 mM reduced glutathione, 50 mM Tris-HCl) and fractions collected until the OD₂₈₀ was 0.1. 21 μl of each fraction were loaded on a 12% SDS gel using either Biorad SDS-PAGE Molecular weight standard broad range (M1) (200, 116.25, 97.4, 66.2, 45, 31, 21.5, 14.4, 6.5 kDa) or Amersham Rainbow Marker (M2) (220, 66, 46, 30, 21.5, 14.3 kDa) as standards. As the MW of GST is 26 kDa, this value must be added to the MW of each GST-fusion protein.

I) His-Fusion Solubility Analysis

To analyse the solubility of the His-fusion expression products, pellets of 3 ml cultures were resuspended in buffer M1 [500 μl PBS pH 7.2]. 25 μl lysozyme (10 mg/ml) was added and the bacteria were incubated for 15 min at 4° C. The pellets were sonicated for 30 sec at 40 W using a Branson sonifier B-15, frozen and thawed twice and then separated again into pellet and supernatant by a centrifugation step. The supernatant was collected and the pellet was resuspended in buffer M2 [8M urea, 0.5M NaCl, 20 mM imidazole and 0.1M NaH₂ PO₄] and incubated for 3 to 4 hours at 4° C. After centrifugation, the supernatant was collected and the pellet was resuspended in buffer M3 [6M guanidinium-HCl, 0.5M NaCl, 20 mM imidazole and 0.1M NaH₂PO₄] overnight at 4° C. The supernatants from all steps were analysed by SDS-PAGE.

J) His-Fusion Large-Scale Purification.

A single colony was grown overnight at 37° C. on a LB+Amp agar plate. The bacteria were inoculated into 20 ml of LB+Amp liquid culture and incubated overnight in a water bath shaker. Bacteria were diluted 1:30 into 600 ml fresh medium and allowed to grow at the optimal temperature (20-37° C.) to OD₅₅₀ 0.6-0.8. Protein expression was induced by addition of 1 mM IPTG and the culture further incubated for three hours. The culture was centrifuged at 8000 rpm at 4° C., the supernatant was discarded and the bacterial pellet was resuspended in 7.5 ml of either (i) cold buffer A (300 mM NaCl, 50 mM phosphate buffer, 10 mM imidazole, pH 8) for soluble proteins or (ii) buffer B (urea 8M, 10 mM Tris-HCl, 100 mM phosphate buffer, pH 8.8) for insoluble proteins.

The cells were disrupted by sonication on ice for 30 sec at 40 W using a Branson sonifier B-15, frozen and thawed two times and centrifuged again.

For insoluble proteins, the supernatant was stored at −20° C., while the pellets were resuspended in 2 ml buffer C (6M guanidine hydrochloride, 100 mM phosphate buffer, 10 mM Tris-HCl, pH 7.5) and treated in a homogenizer for 10 cycles. The product was centrifuged at 13000 rpm for 40 minutes.

Supernatants were collected and mixed with 150 μl Ni²⁺-resin (Pharmacia) (previously washed with either buffer A or buffer B, as appropriate) and incubated at room temperature with gentle agitation for 30 minutes. The sample was centrifuged at 700 g for 5 minutes at 4° C. The resin was washed twice with 10 ml buffer A or B for 10 minutes, resuspended in 1 ml buffer A or B and loaded on a disposable column. The resin was washed at either (i) 4° C. with 2 ml cold buffer A or (ii) room temperature with 2 ml buffer B, until the flow-through reached OD₂₈₀ of 0.02-0.06.

The resin was washed with either (i) 2 ml cold 20 mM imidazole buffer (300 mM NaCl, 50 mM phosphate buffer, 20 mM imidazole, pH 8) or (ii) buffer D (urea 8M, 10 mM Tris-HCl, 100 mM phosphate buffer, pH 6.3) until the flow-through reached the O.D₂₈₀ of 0.02-0.06. The His-fusion protein was eluted by addition of 700 μl of either (i) cold elution buffer A (300 mM NaCl, 50 mM phosphate buffer, 250 mM imidazole, pH 8) or (ii) elution buffer B (urea 8M, 10 mM Tris-HCl, 100 mM phosphate buffer, pH 4.5) and fractions collected until the O.D₂₈₀ was 0.1. 21 μl of each fraction were loaded on a 12% SDS gel.

K) His-Fusion Proteins Renaturation

10% glycerol was added to the denatured proteins. The proteins were then diluted to 20 μg/ml using dialysis buffer I (10% glycerol, 0.5M arginine, 50 mM phosphate buffer, 5 mM reduced glutathione, 0.5 mM oxidised glutathione, 2M urea, pH 8.8) and dialysed against the same buffer at 4° C. for 12-14 hours. The protein was further dialysed against dialysis buffer II (10% glycerol, 0.5M arginine, 50 mM phosphate buffer, 5 mM reduced glutathione, 0.5 mM oxidised glutathione, pH 8.8) for 12-14 hours at 4° C. Protein concentration was evaluated using the formula: Protein (mg/ml)=(1.55×OD ₂₈₀)−(0.76×OD ₂₆₀) L) His-Fusion Large-Scale Purification

500 ml of bacterial cultures were induced and the fusion proteins were obtained soluble in buffer M1, M2 or M3 using the procedure described above. The crude extract of the bacteria was loaded onto a Ni-NTA superflow column (Qiagen) equilibrated with buffer M1, M2 or M3 depending on the solubilization buffer of the fusion proteins. Unbound material was eluted by washing the column with the same buffer. The specific protein was eluted with the corresponding buffer containing 500 mM imidazole and dialysed against the corresponding buffer without imidazole. After each run the columns were sanitized by washing with at least two column volumes of 0.5 M sodium hydroxide and reequilibrated before the next use.

M) Mice Immunisations

20 μg of each purified protein were used to immunise mice intraperitoneally. In the case of ORF 44, CD1 mice were immunised with Al(OH)₃ as adjuvant on days 1, 21 and 42, and immune response was monitored in samples taken on day 56. For ORF 40, CD1 mice were immunised using Freund's adjuvant, rather than Al(OH)₃, and the same immunisation protocol was used, except that the immune response was measured on day 42, rather than 56. Similarly, for ORF 38, CD1 mice were immunised with Freund's adjuvant, but the immune response was measured on day 49.

N) ELISA Assay (Sera Analysis)

The acapsulated MenB M7 strain was plated on chocolate agar plates and incubated overnight at 37° C. Bacterial colonies were collected from the agar plates using a sterile dracon swab and inoculated into 7 ml of Mueller-Hinton Broth (Difco) containing 0.25% Glucose. Bacterial growth was monitored every 30 minutes by following OD₆₂₀. The bacteria were let to grow until the OD reached the value of 0.3-0.4. The culture was centrifuged for 10 minutes at 10000 rpm. The supernatant was discarded and bacteria were washed once with PBS, resuspended in PBS containing 0.025% formaldehyde, and incubated for 2 hours at room temperature and then overnight at 4° C. with stirring. 100 μl bacterial cells were added to each well of a 96 well Greiner plate and incubated overnight at 4° C. The wells were then washed three times with PBT washing buffer (0.1% Tween-20 in PBS). 200 μl of saturation buffer (2.7% Polyvinylpyrrolidone 10 in water) was added to each well and the plates incubated for 2 hours at 37° C. Wells were washed three times with PBT. 200 μl of diluted sera (Dilution buffer: 1% BSA, 0.1% Tween-20, 0.1% NaN₃ in PBS) were added to each well and the plates incubated for 90 minutes at 37° C. Wells were washed three times with PBT. 100 μl of HRP-conjugated rabbit anti-mouse (Dako) serum diluted 1:2000 in dilution buffer were added to each well and the plates were incubated for 90 minutes at 37° C. Wells were washed three times with PBT buffer. 100 μl of substrate buffer for HRP (25 ml of citrate buffer pH5, 10 mg of O-phenildiamine and 10 μl of H₂O) were added to each well and the plates were left at room temperature for 20 minutes. 100 μl H₂SO₄ was added to each well and OD₄₉₀ was followed. The ELISA was considered positive when OD₄₉₀ was 2.5 times the respective pre-immune sera.

O) FACScan Bacteria Binding Assay Procedure.

The acapsulated MenB M7 strain was plated on chocolate agar plates and incubated overnight at 37° C. Bacterial colonies were collected from the agar plates using a sterile dracon swab and inoculated into 4 tubes containing 8 ml each Mueller-Hinton Broth (Difco) containing 0.25% glucose. Bacterial growth was monitored every 30 minutes by following OD₆₂₀. The bacteria were let to grow until the OD reached the value of 0.35-0.5. The culture was centrifuged for 10 minutes at 4000 rpm. The supernatant was discarded and the pellet was resuspended in blocking buffer (1% BSA, 0.4% NaN₃) and centrifuged for 5 minutes at 4000 rpm. Cells were resuspended in blocking buffer to reach OD₆₂₀ of 0.07. 100 μl bacterial cells were added to each well of a Costar 96 well plate. 100 μl of diluted (1:200) sera (in blocking buffer) were added to each well and plates incubated for 2 hours at 4° C. Cells were centrifuged for 5 minutes at 4000 rpm, the supernatant aspirated and cells washed by addition of 200 μl/well of blocking buffer in each well. 100 μl of R-Phicoerytin conjugated F(ab)₂ goat anti-mouse, diluted 1:100, was added to each well and plates incubated for 1 hour at 4° C. Cells were spun down by centrifugation at 4000 rpm for 5 minutes and washed by addition of 200 μl/well of blocking buffer. The supernatant was aspirated and cells resuspended in 200 μl/well of PBS, 0.25% formaldehyde. Samples were transferred to FACScan tubes and read. The condition for FACScan setting were: FL1 on, FL2 and FL3 off; FSC-H threshold: 92; FSC PMT Voltage: E 02; SSC PMT: 474; Amp. Gains 7.1; FL-2 PMT: 539; compensation values: 0.

P) OMV Preparations

Bacteria were grown overnight on 5 GC plates, harvested with a loop and resuspended in 10 ml 20 mM Tris-HCl. Heat inactivation was performed at 56° C. for 30 minutes and the bacteria disrupted by sonication for 10 minutes on ice (50% duty cycle, 50% output). Unbroken cells were removed by centrifugation at 5000 g for 10 minutes and the total cell envelope fraction recovered by centrifugation at 50000 g at 4° C. for 75 minutes. To extract cytoplasmic membrane proteins from the crude outer membranes, the whole fraction was resuspended in 2% sarkosyl (Sigma) and incubated at room temperature for 20 minutes. The suspension was centrifuged at 10000 g for 10 minutes to remove aggregates, and the supernatant further ultracentrifuged at 50000 g for 75 minutes to pellet the outer membranes. The outer membranes were resuspended in 10 mM Tris-HCl, pH8 and the protein concentration measured by the Bio-Rad Protein assay, using BSA as a standard.

Q) Whole Extracts Preparation

Bacteria were grown overnight on a GC plate, harvested with a loop and resuspended in 1 ml of 20 mM Tris-HCl. Heat inactivation was performed at 56° C. for 30 minutes.

R) Western Blotting

Purified proteins (500 ng/lane), outer membrane vesicles (5 μg) and total cell extracts (25 μg) derived from MenB strain 2996 were loaded on 15% SDS-PAGE and transferred to a nitrocellulose membrane. The transfer was performed for 2 hours at 150 mA at 4° C., in transferring buffer (0.3% Tris base, 1.44% glycine, 20% methanol). The membrane was saturated by overnight incubation at 4° C. in saturation buffer (10% skimmed milk, 0.1% Triton X100 in PBS). The membrane was washed twice with washing buffer (3% skimmed milk, 0.1% Triton X100 in PBS) and incubated for 2 hours at 37° C. with mice sera diluted 1:200 in washing buffer. The membrane was washed twice and incubated for 90 minutes with a 1:2000 dilution of horseradish peroxidase labelled anti-mouse Ig. The membrane was washed twice with 0.1% Triton X100 in PBS and developed with the Opti-4CN Substrate Kit (Bio-Rad). The reaction was stopped by adding water.

S) Bactericidal Assay

MC58 strain was grown overnight at 37° C. on chocolate agar plates. 5-7 colonies were collected and used to inoculate 7 ml Mueller-Hinton broth. The suspension was incubated at 37° C. on a nutator and let to grow until OD₆₂₀ was 0.5-0.8. The culture was aliquoted into sterile 1.5 ml Eppendorf tubes and centrifuged for 20 minutes at maximum speed in a microfuge. The pellet was washed once in Gey's buffer (Gibco) and resuspended in the same buffer to an OD₆₂₀ of 0.5, diluted 1:20000 in Gey's buffer and stored at 25° C.

50 μl of Gey's buffer/1% BSA was added to each well of a 96-well tissue culture plate. 25 μl of diluted mice sera (1:100 in Gey's buffer/0.2% BSA) were added to each well and the plate incubated at 4° C. 25 μl of the previously described bacterial suspension were added to each well. 25 μl of either heat-inactivated (56° C. waterbath for 30 minutes) or normal baby rabbit complement were added to each well. Immediately after the addition of the baby rabbit complement, 22 μl of each sample/well were plated on Mueller-Hinton agar plates (time 0). The 96-well plate was incubated for 1 hour at 37° C. with rotation and then 22 μl of each sample/well were plated on Mueller-Hinton agar plates (time 1). After overnight incubation the colonies corresponding to time 0 and time 1 hour were counted.

Table II gives a summary of the cloning, expression and purification results. TABLE II Cloning, expression and purification His-fusion GST-fusion ORF PCR/cloning expression expression Purification orf 38 + + + His-fusion orf 40 + + + His-fusion orf 41 + n.d. n.d. orf 44 + + + His-fusion orf 51 + n.d. n.d. orf 52 + n.d. + GST-fusion orf 56 + n.d. n.d. orf 69 + n.d. n.d. orf 82 + n.d. n.d. orf 114 + n.d. + GST-fusion orf 124 + n.d. n.d.

Example 1

The following partial DNA sequence was identified in N. meningitidis <SEQ ID 1>: 1 ACACTGTTGT TTGCAACGGT TCAGGCAAGT GCTAACCAAT GAAGAGCAAG 51 AAGAAGATTT ATATTTAGAC CCCGTACAAC GCACTGTTGC CGTGTTGATA 101 GTCAATTCCG ATAAAGAAGG CACGGGAGAA AAAGAAAAAG TAGAAGAAAA 151 TTCAGATTGG GCAGTATATT TCAACGAGAA AGGAGTACTA ACAGCCAGAG 201 AAATCACCyT CAAAGCCGGC GACAACCTGA AAATCAAACA AAACGGCACA 251 AACTTCACCT ACTCGCTGAA AAALGACCTC ACAGATCTGA CCAGTGTTGG 301 AACTGAAAAA TTATCGTTTA GCGCAAACGG CAATAAAGTC AACATCACAA 351 GCGACACCAA AGGCTTGAAT TTTGCGAAAG AAACGGCTGG sACGAACGgC 401 GACACCACGG TTCATCTGAA CGGTATTGGT TCGACTTTGA CCGATACGCT 451 GCTGAATACC GGAGCGACCA CAAACGTAAC CAACGACAAC GTTACCGATG 501 ACGAGAAAAA ACGTGCGGCA AGCGTTAAAG ACGTATTAAA CGCTGGCTGG 551 AACATTAAAG GCGTTAAACC CGGTACAACA GCTTCCGATA ACGTTGATTT 601 CGTCCGCACT TACGACACAG TCGAGTTCTT GAGCGCAGAT ACGAAAACAA 651 CGACTGTTAA TGTGGAAAGC AAAGACAACG GCAAGAAAAC CGAAGTTAAA 701 ATCGGTGCGA AGACTTCTGT TATTAAAGAA AAAGAC...

This corresponds to the amino acid sequence <SEQ ID 2; ORF40>: 1 ..TLLFATVQAS ANQEEQEEDL YLDPVQRTVA VLIVNSDKEG TGEKEKVEEN 51   SDWAVYFNEK GVLTAREITX KAGDNLKIKQ NGTNFTYSLK KDLTDLTSVG 101   TEKLSFSANG NKVNITSDTK GLNFAKETAG TNGDTTVHLN GIGSTLTDTL 151   LNTGATTNVT NDNVTDDEKK RAASVKDVLN AGWNIKGVKP GTTASDNVDF 201   VRTYDTVEFL SADTKTITVN VESKDNGKKT EVKIGAXTSV IKEKD...

Further work revealed the complete DNA sequence <SEQ ID 3>: 1 ATGAACAAAA TATACCGCAT CATTTGGAAT AGTGCCCTCA ATGCCTGGGT 51 CGTCGTATCC GAGCTCACAC GCAACCACAC CAAACGCGCC TCCGCAACCG 101 TGAAGACCGC CGTATTGGCG ACACTGTTGT TTGCAACGGT TCAGGCAAGT 151 GCTAACAATG AAGAGCAAGA AGAAGATTTA TATTTAGACC CCGTACAACG 201 CACTGTTGCC GTGTTGATAG TCAATTCCGA TAAAGAAGGC ACGGGAGAAA 251 AAGAAAAAGT AGAAGAAAAT TCAGATTGGG CAGTATATTT CAACGAGAAA 301 GGAGTACTAA CAGCCAGAGA AATCACCCTC AAAGCCGGCG ACAACCTGAA 351 AATCAAACAA AACGGCACAA ACTTCACCTA CTCGCTGAAA AAAGACCTCA 401 CAGATCTGAC CAGTGTTGGA ACTGAAAAAT TATCGTTTAG CGCAAACGGC 451 AATAAAGTCA ACATCACAAG CGACACCAAA GGCTTGAATT TTGCGAAAGA 501 AACGGCTGGG ACGAACGGCG ACACCACGGT TCATCTGAAC GGTATTGGTT 551 CGACTTTGAC CGATACGCTG CTGAATACCG GAGCGACCAC AAACGTAACC 601 AACGACAACG TTACCGATGA CGAGAAAAAA CGTGCGGCAA GCGTTAAAGA 651 CGTATTAAAC GCTGGCTGGA ACATTAAAGG CGTTAAACCC GGTACAACAG 701 CTTCCGATAA CGTTGATTTC GTCCGCACTT ACGACACAGT CGAGTTCTTG 751 AGCGCAGATA CGAAAACAAC GACTGTTAAT GTGGAAAGCA AAGACAACGG 801 CAAGAAAACC GAAGTTAAAA TCGGTGCGAA GACTTCTGTT ATTAAAGAAA 851 AAGACGGTAA GTTGGTTACT GGTAAAGACA AAGGCGAGAA TGGTTCTTCT 901 ACAGACGAAG GCGAAGGCTT AGTGACTGCA AAAGAAGTGA TTGATGCAGT 951 AAACAAGGCT GGTTGGAGAA TGAAAACAAC AACCGCTAAT GGTCAAACAG 1001 GTCAAGCTGA CAAGTTTGAA ACCGTTACAT CAGGCACAAA TGTAACCTTT 1051 GCTAGTGGTA AAGGTACAAC TGCGACTGTA AGTAAAGATG ATCAAGGCAA 1101 CATCACTGTT ATGTATGATG TAAATGTCGG CGATGCCCTA AACGTCAATC 1151 AGCTGCAAAA CAGCGGTTGG AATTTGGATT CCAAAGCGGT TGCAGGTTCT 1201 TCGGGCAAAG TCATCAGCGG CAATGTTTCG CCGAGCAAGG GAAAGATGGA 1251 TGAAACCGTC AACATTAATG CCGGCAACAA CATCGAGATT ACCCGCAACG 1301 GTAAAAATAT CGACATCGCC ACTTCGATGA CCCCGCAGTT TTCCAGCGTT 1351 TCGCTCGGCG CGGGGGCGGA TGCGCCCACT TTGAGCGTGG ATGGGGACGC 1401 ATTGAATGTC GGCAGCAAGA AGGACAACAA ACCCGTCCGC ATTACCAATG 1451 TCGCCCCGGG CGTTAAAGAG GGGGATGTTA CAAACGTCGC ACAACTTAAA 1501 GGCGTGGCGC AAAACTTGAA CAACCGCATC GACAATGTGG ACGGCAACGC 1551 GCGTGCGGGC ATCGCCCAAG CGATTGCAAC CGCAGGTCTG GTTCAGGCGT 1601 ATTTGCCCGG CAAGAGTATG ATGGCGATCG GCGGCGGCAC TTATCGCGGC 1651 GAAGCCGGTT ACGCCATCGG CTACTCCAGT ATTTCCGACG GCGGAAATTG 1701 GATTATCAAA GGCACGGCTT CCGGCAATTC GCGCGGCCAT TTCGGTGCTT 1751 CCGCATCTGT CGGTTATCAG TGGTAA

This corresponds to the amino acid sequence <SEQ ID 4; ORF40-1>: 1 MNKIYRIIWN SALNAWVVVS ELTRNHTKRA SATVKTAVLA TLLFATVQAS 51 ANNEEQEEDL YLDFVQRTVA VLIVNSDKEG TGEKEKVEEN SDWAVYFNEK 101 GVLTAREITL KAGDNLKIKQ NGTNFTYSLK KDLTDLTSVG TEKLSFSMIG 151 NKVNITSDTK GLNFAKETAG TNGDTTVHLN GIGSTLTDTL LNTGATTNVT 201 NDNVTDDEKK RAASVKDVLN AGWNIKGVKP GTTASDNVDF VRTYDTVEFL 251 SADTKTTTVN VESKDNGKKT EVKIGAKTSV IKEKDGKLVT GKDKGENGSS 301 TDEGEGLVTA KEVIDAYNKA GWRMKTTTAN GQTGQADKFE TVTSGTNVTF 351 ASGKGTTATV SKDDQGNITV NYDVNVGDAL NVNQLQNSGW NLDSKAVAGS 401 SGKVISGNVS PSKGKMDETV NINAGNNIEI TRNGKNIDIA TSHTPQFSSV 451 SLGAGADAPT LSVDGDALNV GSKKDNKPVR ITNVAPGVKE GOVTNVAQLK 501 GVAQNLNNRI DNVDGNARAG ZAQAIATAGL VQAYLPGKSM MAIGGGTYRG 551 EAGYAIGYSS ISDGGNWIIK GTASGNSRGH FGASASVGYQ W*

Further work identified the corresponding gene in strain A of N. meningitidis <SEQ ID 5>: 1 ATGAACAAAA TATACCGCAT CATTTGGAAT AGTGCCCTCA ATGCCTGNGT 51 CGCCGTATCC GAGCTCACAC GCAACCACAC CAAACGCGCC TCCGCAACCG 101 TGAAGACCGC CGTATTGGCG ACACTGTTGT TTGCAACGGT TCAGGCGAAT 151 GCTACCGATG AAGATGAAGA AGAAGAGTTA GAATCCGTAC AACGCTCTGT 201 CGTAGGGAGC ATTCAAGCCA GTATGGAAGG CAGCGGCGAA TTGGAAACGA 251 TATCATTATC AATGACTAAC GACAGCAAGG AATTTGTAGA CCCATACATA 301 GTAGTTACCC TCAAAGCCGG CGACAACCTG AAAATCAAAC AAAACACCAA 351 TGAAAACACC AATGCCAGTA GCTTCACCTA CTCGCTGAAA AAAGACCTCA 401 CAGGCCTGAT CAATGTTGAN ACTGAAAAAT TATCGTTTGG CGCAAACGGC 451 AAGAAAGTCA ACATCATAAG CGACACCAAA GGCTTGAATT TCGCGAAAGA 501 AACGGCTGGG ACGAACGGCG ACACCACGGT TCATCTGAAC GGTATCGGTT 551 CGACTTTGAC CGATACGCTT GCGGGTTCTT CTGCTTCTCA CGTTGATGCG 601 GGTAACCNAA GTACACATTA CACTCGTGCA GCAAGTATTA AGGATGTGTT 651 GAATGCGGGT TGGAATATTA AGGGTGTTAA ANNNGGCTCA ACAACTGGTC 701 AATCAGAAAA TGTCGATTTC GTCCGCACTT ACGACACAGT CGAGTTCTTG 751 AGCGCAGATA CGNAAACAAC GACNGTTAAT GTGGAAAGCA AAGACAACGG 801 CAAGAGAACC GAAGTTAAAA TCGGTGCGAA GACTTCTGTT ATTAAAGAAA 851 AAGACGGTAA GTTGGTTACT GGTAAAGGCA AAGGCGAGAA TGGTTCTTCT 901 ACAGACGAAG GCGAAGGCTT AGTGACTGCA AAAGAAGTGA TTGATGCAGT 951 AAACAAGGCT GGTTGGAGAA TGAAAACAAC AACCGCTAAT GGTCAAACAG 1001 GTCAAGCTGA CAAGTTTGAA ACCGTTACAT CAGGCACAAA TGTAACCTTT 1051 GCTAGTGGTA AAGGTACAAC TGCGACTGTA AGTAAAGATG ATCAAGGCAA 1101 CATCACTGTT ATGTATGATG TAAATGTCGG CGATGCCCTA AACGTCAATC 1151 AGCTGCAAAA CAGCGGTTGG AATTTGGATT CCAAAGCGGT TGCAGGTTCT 1201 TCGGGCAAAG TCATCAGCGG CAATGTTTCG CCGAGCAAGG GAAAGATGGA 1251 TGAAACCGTC AACATTAATG CCGGCAACAA CATCGACATT AGCCGCAACG 1301 GTAAAAATAT CGACATCGCC ACTTCGATGG CGCCGCAGTT TTCCAGCGTT 1351 TCGCTCGGCG CGGGGGCAGA TGCGCCCACT TTAAGCGTGG ATGACGAGGG 1401 CGCGTTGAAT GTCGGCAGCA AGGATGCCAA CAAACCCGTC CGCATTACCA 1451 ATGTCGCCCC GGGCGTTAAA GANGGGGATG TTACAAACGT CNCACAACTT 1501 AAAGGCGTGG CGCAAAACTT GAACAACCGC ATCGACAATG TGGACGGCAA 1551 CGCGCGTGCN GGCATCGCCC AAGCGATTGC AACCGCAGGT CTGGTTCAGG 1601 CGTATCTGCC CGGCAAGAGT ATGATGGCGA TCGGCGGCGG CACTTATCGC 1651 GGCGAAGCCG GTTACGCCAT CGGCTACTCC AGTATTTCCG ACGGCGGAAA 1701 TTGGATTATC AAAGGCACGG CTTCCGGCAA TTCGCGCGGC CATTTCGGTG 1751 CTTCCGCATC TGTCGGTTAT CAGTGGTAA

This encodes a protein having amino acid sequence <SEQ ID 6; ORF40a>: 1 MNKIYRIIWN SALNPXVAVS ELTRNHTKRA SATVKTAVLA TLLFATVQAN 51 ATDEDEKEEL ESVQRSVVGS IQASMEGSGE LETISLSHTN DSKEFVDPYI 101 VVTLKAGDNL KIKONTHENT NASSFTYSLK KDLTGLINVX TEKLSFGANG 151 KKVNIISDTK GLNFAXETAG TNGDTTVHLN GIGSTLTDTL AGSSASHVDA 201 GNXSTHYTRA ASIKDVLNAG WNIKGVKXGS TTGQSENVDF VRTYDTVEFL 251 SADTXTTTVN VESKDNGKRT EVXIGAXTSV IKEKDGKLVT GKGKGENGSS 301 TDEGEGLVTA KEVIDAVNKA GWRMKTTTAN GQTGQADKFE TVTSGTNVTF 351 ASGKGTTATV SXDDQGNITV MYDVNVGDAL NVNQLONSGW NLDSKAVAGS 401 SGKVISGNVS PSKGKMDETV NINAGNNIEI SRNGKNIDIA TSMAPQFSSV 451 SLGAGADAPT LSVDDEGALN VGSKDANKPV RITNVAPGVK XGDVTNVXQL 501 KGVAQNLNNR IDNYOGNARA GIAQAIATAG LVQAYLPGKS NNAIGGGTYR 551 GEAGYAIGYS SISDGGNWII KGTASGNSRG HFGASASVGY QW

The originally-identified partial strain B sequence (ORF40) shows 65.7% identity over a 254 aa overlap with ORF40a:                                      10        20        30 orf40.pep                              TLLFATVQASANQEEQEEDLYLDPVQRTVA                              |||||||||:|::|::||:|  : |||:| orf40a SALNAXVAVSELTRNHTKRASATVKTAVLATLLFATVQANATDEDEEEEL--ESVQRSV-         20        30        40        50        60         40        50        60        70        80 orf40.pep VLIVNSDKEGTGEKEKVEEN-SDWAVYFNEKGVLTAREITXKAGDNLKIKQN------GT |  :::: ||:|| | :  : :: :  | :  ::    :| |||||||||||      :: orf40a VGSIQASMEGSGELETISLSMTNDSKEFVDPYIV----VTLKAGDNLKIKQNTNENTNAS  70        80        90       100       110       120      90       100       110       120       130       140 orf40.pep NFTYSLKKDLTDLTSVGTEKLSFSANGNKVNITSDTKGLNFAKETAGTNGDTTVHLNGIG :|||||||||| | :| ||||||:|||:|||| ||||||||||||||||||||||||||| orf40a SFTYSLKKDLTGLINVXTEKLSFGANGKKVNIISDTKGLNFAKETAGTNGDTTVHLNGIG     130       140       150       160       170       180     150       160       170       180       190       200 orf40.pep STLTDTLLNTGATTNVTNDNVTDDEKKRAASVKDVLNAGWNIKGVKPGTTA--SDNVDFV ||||||| :::|: :|   | :  :  ||||:|||||||||||||| |:|:  |:||||| orf40a STLTDTLAGSSAS-HVDAGNXST-HYTRAASIKDVLNAGWNIKGVKXGSTTGQSENVDFV     190       200       210       220       230       240       210       220       230       240 orf40.pep RTYDTVEFLSADTKTTTVNVESKDNGKKTEVKIGAKTSVIKEKD ||||||||||||| |||||||||||||:|||||||||||||||| orf40a RTYDTVEFLSADTXTTTVNVESKDNGKRTEVKIGAKTSVIKEKDGKLVTGKGKGENGSST       250       260       270       280       290       300

The complete strain B sequence (ORF44-1) and ORF40a show 83.7% identity in 601 aa overlap:           10        20        30        40        50        60 orf40-1.pep   MNKIYRIIWNSALNAWVVVSELTRNHTKRASATVKTAVLATLLFATVQASANNEEQEEDL   ||||||||||||||| |:|||||||||||||||||||||||||||||||:|::|::||:| orf40a   MNKIYRIIWNSALNAXVAVSELTRNHTKRASATVKTAVLATLLFATVQANATDEDEEEEL           10        20        30        40        50        60           70        80        90       100       110       119 orf40-1.pep   YLDPVQRTVAVLIVNSDKEGTGEKEKVEEN-SDWAVYFNEKGVLTAREITLKAGDNLKIK     : |||:| |  :::: ||:|| | :  : :: :  | :  ::    :||||||||||| orf40a   --ESVQRSV-VGSIQASMEGSGELETISLSMTNDSKEFVDPYIV----VTLKAGDNLKIK              70        80        90       100       110 120             130       140       150       160       170 orf40-1.pep   QN------GTNFTYSLKKDLTDLTSVGTEKLSFSANGNKVNITSDTKGLNFAKETAGTNG   ||      :::||||||||| | :| ||||||:|||:|||| ||||||||||||||||| orf40a   QNTNENTNASSFTYSLKKDLTGLINVXTEKLSFGANGKKVNIISDTKGLNFAKETAGTNG       120       130       140       150       160       170       180       190       200       210       220       230 orf40-1.pep   DTTVHLNGIGSTLTDTLLNTGATTNVTNDNVTDDEKKRAASVKDVLNAGWNIKGVKPGTT   ||||||||||||||||| :::|: :|   | :  :  ||||:|||||||||||||| |:| orf40a   DTTVHLNGIGSTLTDTLAGSSAS-HVDAGNXST-HYTRAASIKDVLNAGWNIKGVKXGST       180       190       200       210       220       230         240       250       260       270       280       290 orf40-1.pep   A--SDNVDFVRTYDTVEFLSADTKTTTVNVESKDNGKKTEVKIGAKTSVIKEKDGKLVTG   :  |:|||||||||||||||||| |||||||||||||:|||||||||||||||||||||| orf40a   TGQSENVDFVRTYDTVEFLSADTXTTTVNVESKDNGKRTEVKIGAKTSVIKEKDGKLVTG         240       250       260       270       280       290         300       310       320       330       340       350 orf40-1.pep   KDKGENGSSTDEGEGLVTAKEVIDAVNKAGWRMKTTTANGQTGQADKFETVTSGTNVTFA   | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||| orf40a   KGKGENGSSTDEGEGLVTAKEVIDAVNKAGWRMKTTTANGQTGQADKFETVTSGTNVTFA         300       310       320       330       340       350         360       370       360       390       400       410 orf40-1.pep   SGKGTTATVSKDDQGNITVMYDVNVGDALNVNQLQNSGWNLDSKAVAGSSGKVISGNVSP   |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| orf40a   SGKGTTATVSKDDQGNITVMYDVNVGDALNVNQLQNSGWNLDSKAVAGSSGKVISGNVSP         360       370       360       390       400       410         420       430       440       450       460       470 orf40-1.pep   SKGKMDETVNINAGNNIEITRNGKNIDIATSMTPQFSSVSLGAGADAPTLSVDGD-ALNV   |||||||||||||||||||:||||||||||||:|||||||||||||||||||| : |||| orf40a   SKGKMDETVNINAGNNIEISRNGKNIDIATSMAPQFSSVSLGAGADAPTLSVDDEGALNV         420       430       440       450       460       470          480       490       500       510       520       530 orf40-1.pep   GSKKDNKPVRITNVAPGVKEGDVTNVAQLKGVAQNLNNRIDNVDGNARAGIAQAIATAGL   |||  |||||||||||||| |||||| ||||||||||||||||||||||||||||||||| orf40a   GSKDANKPVRITNVAPGVKXGDVTNVXQLKGVAQNLNNRIDNVDGNARAGIAQAIATAGL       480       490       500       510       520       530          540       550       560       570       580       590 orf40-1.pep   VQAYLPGKSMMAIGGGTYRGEAGYAIGYSSISDGGNWIIKGTASGNSRGHFGASASVGYQ   |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| orf40a   VQAYLPGKSMMAIGGGTYRGEAGYAIGYSSISDGGNWIIKGTASGNSRGHFGASASVGYQ          540       550       560       570       580       590 orf40-1.pep   WX   || orf40a   WX

Computer analysis of these amino acid sequences gave the following results:

Homology with Hsf Protein Encoded by the Type b Surface Fibrils Locus of H. influenzae (Accession Number U41852)

ORF40 and Hsf protein show 54% aa identity in 251 aa overlap: Orf40   1 TLLFATVQASANQEEQEEDLYLDPVQRTVAVLVINSDXXXXXXXXXXXXNSDWAVYFNEK 60     TLLFATVQA+A  E++E    LDPV RT  VL  +SD            NS+W +YF+ K Hsf   41 TLLFATVQANATDEDEE----LDPVVRTAPVLSFHSDKEGTGEKEVTE-NSNWGIYFDNK 95 Orf40  61 GVLTAREITXKAGDNLKIKQN------GTNFTYSLKKDLTDLTSVGTEKLSFSANGNKVN 114     GVL A  IT KAGDNLKIKQN       ++FTYSLKKDLTDLTSV TEKLSF ANG+KV+ Hsf  96 GVLKAGAITLKAGDNLKIKQNTDESTNASSFTYSLKKDLTDLTSVATEKLSFGANGDKVD 155 Orf40 115 ITSDTKGLNFAKETAGTNGDTTVhLNGIGSTLTDTLLNTGAXXXXXXXXXXXXEKKRAAS 174     ITSD  GL  AK      G+  VHLNG+ STL D + NTG             EK RAA+ Hsf 156 ITSDANGLKLAK-----TGNGNVHLNGLDSTLPDAVTNTGVLSSSSFTPNDV-EKTRAAT 209 Orf40 175 VKDVLNAGWNIKGVKPGTTASDNVDFVRTYDTVEFLSADTKTTTVNVESKDNGKKTEVKI 234     VKDVLNAGWNIKG K      ++VD V  Y+ VEF++ D  T  V ++K+NGK TEVK Hsf 210 VKDVLNAGWNIKGAKTAGGNVESVDLVSAYNNVEFITGDKNTLDVVLTAKENGKTTEVKF 269 Orf40 235 GAKTSVIKEKD 245       KTSVIKEKD Hsf 270 TPKTSVIKEKD 280

ORF40a also shows homology to Hsf: gi|1666683 (U41852) hsf gene product [Haemophilus influenzae] Length = 2353 Score = 153 (67.7 bits), Expect = 1.5−116, Sum P(11) = 1.5e−116 Identities = 33/36 (91%), Positives = 34/36 (94%) Query:   16 VAVSELTRNHTKRASATVKTAVLATLLFATVQANAT 51             V VSELTR HTKRASATV+TAVLATLLFATVQNAT Sbjct:   17 VVVSELTRTHTKRASATVETAVLATLLFATVQANAT 52 Score = 161 (71.2 bits), Expect = 1.5e−116, Sum P(11) 1.5e−116 Identities = 32/38 (84%), Positives = 36/38 (94%) Query:  101 VTLKAGDNLKIKQNTNENTNASSFTYSLKKDLTGLINV 138             +TLAGDNLKIKQNT+E+TNASSFTYSLKKDLT L +V Sbjct:  103 ITLKAGDNLKIKQNTDESTNASSFFYSLKKDLTDLTSV 140 Score = 110 (48.7 bits), Expect = 1.5e−116, Sum P(11) = 1.5e−116 Identities = 21/29 (72%), Positives = 25/29 (86%) Query:  138 VTEKLSFGANGKKVNIISDTKGLNFAKET 166             V++KLS G NG KVNI SDTKGLNFAK++ Sbjct: 1439 VSDKLSLGTNGNKVNITSDTXGLNFAKDS 1467 Score = 85 (37.6 bits), Expect = 1.5e−116, Sum P(11) = 1.5e−116 Identities = 18/32 (56%), Positives = 20/32 (62%) Query:  169 TNGDTTVHLNGIGSTLTDTLAGSSASHVDAGN 200             T  D  +HLNGI STLTDTL  S A+    GN Sbjct: 1469 TGDDANIHLNGIASTLTDTLLNSGATTNLGGN 1500 Score = 92 (40.7 bits), Expect = 1.5e−116, Sum P(11) = 1.5e−116 Identities = 16/19 (84%), Positives = 19/19 (100%) Query:  206 RAASIKOVLNAGWNIKGVK 224             RAAS+KDVLNAGWN++GVK Sbjct: 1509 RAASVKDVLNAGWNVRGVK 1527 Score = 90 (39.8 bits), Expect = 1.5e−116, Sum P(11) = 1.5e−116 Identities = 17/28 (60%), Positives 20/28 (71%) Query:  226 STTGQSENVDFVRTYDTVEFLSADTTTT 253             S   Q EN+DFV TYDTV+F+S D  TT Sbjct: 1530 SANNQVENIDFVATYDTVDFVSGDKDTT 1557

Based on homology with Hsf, it was predicted that this protein from N. meningitidis, and its epitopes, could be useful antigens for vaccines or diagnostics.

ORF40-1 (61 kDa) was cloned in pET and pGex vectors and expressed in E. coli, as described above. The products of protein expression and purification were analyzed by SDS-PAGE. FIG. 1A shows the results of affinity purification of the His-fusion protein, and FIG. 1B shows the results of expression of the GST-fusion in E. coli. Purified His-fusion protein was used to immunise mice, whose sera were used for FACS analysis (FIG. 1C), a bactericidal assay (FIG. 1D), and ELISA (positive result). These experiments confirm that ORF40-1 is a surface-exposed protein, and that it is a useful immunogen.

FIG. 1E shows plots of hydrophilicity, antigenic index, and AMPHI regions for ORF40-1.

Example 2

The following partial DNA sequence was identified in N. meningitidis <SEQ ID 7> 1 ATGTFACGTt TGACTGCtTT AGCCGTATGC ACCGCCCTCG CTTTGGGCGC 51 GTGTT~GCCG CAAAATTCCG ACTCTGCCCC ACAAGCCAAA GaACAGGCGG 101 TTTCCGCCGC ACAAACCGAA GgCGCGTCCG TTACCGTCAA AACCGCGCGC 151 GGCGACGTTC AAATACCGCA AAACCCCGAA CGCATCGCCG TTTACGATTT 201 GGGTATGCTC GACACCTTGA GCAAACTGGG CGTGAAAACC GGTTTGTCCG 251 TCGATAAAAA CCGCCTGCCG TATTTAGAGG AATATTTCAA AACGACAAAA 301 CCTGCcGGCA CTTTGTTCGA GCCGGATTAC GAAACGCTCA ACGCTTACAA 351 ACCGCAGCTC ATCATCATCG GCAGCCGCGC CgCCAAGGCG TTTGACAAAT 401 TGAAcGAAAT CGCGCCGACC ATCGrmwTGA CCGCCGATAC CGCCAACCTC 451 AAAGAAAGTG CCAArGAGGC ATCGACGCTG GCGCAAATCT TC..

This corresponds to the amino acid sequence <SEQ ID 8; ORF38>: 1 MLRLTALAVC TALALGACSP QNSDSAPOAK EQAVSAAQTE GASVTVKTAR 51 GDVQIPQNPE RIAVYDLGHL DTLSKLGVKT GLSVDKNRLP YLEEYFKTTK 101 PAGTLFEPDY ETLNAYKPQL IIIGSRAAKA FDKLNEIAPT IXXTADTANL 151 KESAKEASTL AQIF..

Further work revealed the complete nucleotide sequence <SEQ ID 9>: 1 ATGTTACGTT TGACTGCTTT AGCCGTATGC ACCGCCCTCG CTTTGGGCGC 51 GTGTTCGCCG CAAAATTCCG ACTCTGCCCC ACAAGCCAAA GAACAGGCGG 101 TTTCCGCCGC ACAAACCGAA GGCGCGTCCG TTACCGTCAA AACCGCGCGC 151 GGCGACGTTC AAATACCGCA AAACCCCGAA CGCATCGCCG TTTACGATTT 201 GGGTATGCTC GACACCTTGA GCAAACTGGG CGTGAAAACC GGTTTGTCCG 251 TCGATAAAAA CCGCCTGCCG TATTTAGAGG AATATTTCAA AACGACAAAA 301 CCTGCCGGCA CTTTGTTCGA GCCGGATTAC GAAACGCTCA ACGCTTACAA 351 ACCGCAGCTC ATCATCATCG GCAGCCGCGC CGCCAAGGCG TTTGACAAAT 401 TGAACGAAAT CGCGCCGACC ATCGAAATGA CCGCCGATAC CGCCAACCTC 451 AAAGAAAGTG CCAAAGAGCG CATCGACGCG CTGGCGCAAA TCTTCGGCAA 501 ACAGGCGGAA GCCGACAAGC TGAAGGCGGA AATCGACGCG TCTTTTGAAG 551 CCGCGAAAAC TGCCGCACAA GGTAAGGGCA AAGGTTTGGT GATTTTGGTC 601 AACGGCGGCA AGATGTCGGC TTTCGGCCCG TCTTCACGCT TGGGCGGCTG 651 GCTGCACAAA GACATCGGCG TTCCCGCTGT CGATGAATCA ATTAAAGAAG 701 GCAGCCACGG TCAGCCTATC AGCTTTGAAT ACCTGAAAGA GAAAAATCCC 751 GACTGGCTGT TTGTCCTTGA CCGAAGCGCG GCCATCGGCG AAGAGGGTCA 801 GGCGGCGAAA GACGTGTTGG ATAATCCGCT GGTTGCCGAA ACAACCGCTT 851 GGAAAAAAGG ACAGGTCGTG TACCTCGTTC CTGAAACTTA TTTGGCAGCC 901 GGTGGCGCGC AAGAGCTGCT GAATGCAAGC AAACAGGTTG CCGACGCTTT 951 TAACGCGGCA AAATAA

This corresponds to the amino acid sequence <SEQ ID 10; ORF38-1>: 1 MLRLTALAVC TALALGACSP QNSDSAPQAK EQAVSAAQTE GASVTVKTAR 51 GDVQIPQNPE RIAVYDLQIL DTLSXLGVKT GLSVDKNRLP YLEEYFKTTK 101 PAGTLFEPDY ETLNAYKPQL IIIGSRAAKA FDKLNEIAPT IENTADTANL 151 KESAKERIDA LAQIFGKQAE ADKLKAEIDA SFEAAKTAAQ GKGKGLVILV 201 NGGKMSAFGP SSRLGGWLKK DIGVPAVDES IKEGSHGQPI SFEYLKEKNP 251 DWLFVLDRSA AIGEEGQAAK DVLDNPLVAE TTAWKKGQVV YLVPETYLAA 301 GGAQELLNAS KQVADAFNAA K*

Computer analysis of this amino acid sequence reveals a putative prokaryotic membrane lipoprotein lipid attachment site (underlined).

Further work identified the corresponding gene in strain A of N. meningitidis <SEQ ID 11>: 1 ATGTTACGTT TGACTGCTTT AGCCGTATGC ACCGCCCTCG CTTTGGGCGC 51 GTGTTCGCCG CAAAATTCCG ACTCTGCCCC ACAAGCCAAA GAACAGGCGG 101 TTTCCGCCGC ACAATCCGAA GGCGTGTCCG TTACCGTCAA AACGGCGCGC 151 GGCGATGTTC AAATACCGCA AAACCCCGAA CGTATCGCCG TTTACGATTT 201 GGGTATGCTC GACACCTTGA GCAAACTGGG CGTGAAAACC GGTTTGTCCG 251 TCGATAAAAA CCGCCTGCCG TATTTAGAGG AATATTTCAA AACGACAAAA 301 CCTGCCGGAA CTTTGTTCGA GCCGGATTAC GAAACGCTCA ACGCTTACAA 351 ACCGCAGCTC ATCATCATCG GCAGCCGCGC AGCCAAAGCG TTTGACAAAT 401 TGAACGAAAT CGCGCCGACC ATCGAAATGA CCGCCGATAC CGCCAACCTC 451 AAAGAAAGTG CCAAAGAGCG TATCGACGCG CTGGCGCAAA TCTTCGGCAA 501 AAAGGCGGAA GCCGACAAGC TGAAGGCGGA AATCGACGCG TCTTTTGAAG 551 CCGCGAAAAC TGCCGCGCAA GGCAAAGGCA AGGGTTTGGT GATTTTGGTC 601 AAcGGCGGCA AGATGTCCGC CTTCGGCCCG TCTTCACGAC TGGGCGGCTG 651 GCTGCACAAA GACATCGGCG TTCCCGCTGT TGACGAAGCC ATCAAAGAAG 701 GCAGCCACGG TCAGCCTATC AGCTTTGAAT ACCTGAAAGA GAAAAATCCC 751 GACTGGCTGT TTGTCCTTGA CCGCAGCGCG GCCATCGGCG AAAAGGGTCA 601 GGCGGCGAAA GACGTGTTGA ACAATCCGCT GGTTGCCGAA ACAACCGCTT 851 GGAAAAATGG ACAAGTCGTT TACCTTGTTC CTGAAACTTA TTTGGCAGCC 901 GGTGGCGCGC AAGAGCTACT GAATGCAAGC AAACAGGTTG CCGACGCTTT 951 TAACGCGGCA AAATAA

This encodes a protein having amino acid sequence <SEQ ID 12; ORF38a>: 1 MLRLTALAVC TALALGACSP QNSDSAPOAK EQAVSAAQSE GVSVTVKTAR 51 GDVQIPQNPE RIAVYDLGHL DTLSKLGVKT GLSVDKNRLP YLEEYFKTTK 101 PAGTLFEPDY ETLNAYKPQL IIIGSRAAKA FDKLNEIAPT IENTADTANL 151 KESAKERIDA LAOIFGKKAE ADKLKAEIDA SFEAAKTAAQ GKGKGLVILV 201 NGGKMSAFGP SSRLGGWLHK DIGVPAVDEA IKEGSHGQPI SFEYLKEKNP 251 DWLFVLDRSA AIGEEGQAAK DVLNNPLVAE TTAWKKGQVV YLVPETYLAA 301 GGAQELLNAS KQVAOAFWAA K*

The originally-identified partial strain B sequence (ORF38) shows 95.2% identity over a 165 aa overlap with ORF38a:         10        20        30        40        50        60 orf38.pep MLRLTALAVCTALALGACSPQNSDSAPQAKEQAVSAAQTEGASVTVKTARGDVQIPQNPE ||||||||||||||||||||||||||||||||||||||:||:|||||||||||||||||| orf38a MLRLTALAVCTALALGACSPQNSDSAPQAKEQAVSAAQSEGVSVTVKTARGDVQIPQNPE         10        20        30        40        50        60         70        80        90       100       110       120 orf38.pep RIAVYDLGMLDTLSKLGVKTGLSVDKNRLPYLEEYFKTTKPAGTLFEPDYETLNAYKPQL |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| orf38a RIAVYDLGMLDTLSKLGVKTGLSVDKNRLPYLEEYFKTTKPAGTLFEPDYETLNAYKPQL         70        80        90       100       110       120        130       140       150       160 orf38.pep IIIGSRAAKAFDKLNEIAPTIXXTADTANLKESAKE-ASTLAQIF |||||||||||||||||||||  |||||||||||||  ::||||| orf39a IIIGSRAAKAFDKLNEIAPTIEMTADTANLKESAKERIDALAQIFGKKAEADKLKAEIDA        130       140       150       160 orf38a SFEAAKTAAQGKGKGLVILVNGGKMSAFGPSSRLGGWLHKDIGVPAVDEAIKEGSHGQPI        190       200       210       220       230       240

The complete strain B sequence (ORF38-1) and ORF38a show 98.4% identity in 321 aa overlap: orf38a.pep MLRLTALAVCTALALGACSPQNSDSAPQAKEQAVSAAQSEGVSVTVKTARGDVQIPQNPE ||||||||||||||||||||||||||||||||||||||:||:|||||||||||||||||| orf38-1 MLRLTALAVCTALALGACSPQNSDSAPQAKEQAVSAAQTEGASVTVKTARGDVQIPQNPE orf38a.pep RIAVYDLGMLDTLSKLGVKTGLSVDKNRLPYLEEYFKTTKPAGTLFEPDYETLNAYKPQL |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| orf38-1 RIAVYDLGMLDTLSKLGVKTGLSVDKNRLPYLEEYFKTTKPAGTLFEPDYETLNAYKPQL orf38a.pep IIIGSRAAKAFDKLNEIAPTIEMTADTANLKESAKERIDALAQIFGKKAEADKLKAEIDA |||||||||||||||||||||||||||||||||||||||||||||||:|||||||||||| orf38-1 IIIGSRAAKAFDKLNEIAPTIEMTADTANLKESAKERIDALAQIFGKQAEADKLKAEIDA orf38a.pep SFEAAKTAAQGKGKGLVILVNGGKMSAFGPSSRLGGWLHKDIGVPAVDEAIKEGSHGQPI |||||||||||||||||||||||||||||||||||||||||||||||||:|||||||||| orf38-1 SFEAAKTAAQGKGKGLVILVNGGKMSAFGPSSRLGGWLHKDIGVPAVDESIKEGSHGQPI orf38a.pep SFEYLKEKNPDWLFVLDRSAAIGEEGQAAKDVLNNPLVAETTAWKKGQVVYLVPETYLAA |||||||||||||||||||||||||||||||||:|||||||||||||||||||||||||| orf38-1 SFEYLKEKNPDWLFVLDRSAAIGEEGQAAKDVLDNPLVAETTAWKKGQVVYLVPETYLAA orf38a.pep GGAQELLNASKQVADAFNAAK ||||||||||||||||||||| orf38-1 GGAQELLNASKQVADAFNAAK

Computer analysis of these sequences revealed the following:

Homology with a Lipoprotein (lipo) of C. jejuni (Accession Number X82427)

ORF38 and lipo show 38% aa identity in 96 aa overlap: Orf38:  40 EGASVTVKTARGDVQIPQNPERIAVYDLGMLDTLSKLGVKTGLS-VKDNRLPYLEEYFKT 98     EG S  VK  + G+ + P+NP  ++ + DLG+LDT   L +   ++ V    LP   + FK Lipo:  51 EGDSFLVKDSLGENKTPKNPSKVVILDLGILDTFDALKLNDKVAGVPAKNLPKYLQQFKN 110 Orf38:  99 TKPAGTLFEPDYETLNAYKPQLIIIGSRAAKAFDKL 134         G + + D+E +NA KP LIII  R +K +DKL Lipo: 111 KPSVGGVQQVDFEAINALKPDLIIISGRQSKFYDKL 146

Based on this analysis, it was predicted that this protein from N. meningitidis, and its epitopes, could be useful antigens for vaccines or diagnostics.

ORF38-1 (32 kDa) was cloned in pET and pGex vectors and expressed in E. coli, as described above. The products of protein expression and purification were analyzed by SDS-PAGE. FIG. 2A shows the results of affinity purification of the His-fusion protein, and FIG. 2B shows the results of expression of the GST-fusion in E. coli. Purified His-fusion protein was used to immunise mice, whose sera were used for Western blot analysis (FIG. 2C) and FACS analysis (FIG. 2D). These experiments confirm that ORF38-1 is a surface-exposed protein, and that it is a useful immunogen.

FIG. 2E shows plots of hydrophilicity, antigenic index, and AMPHI regions for ORF38-1.

Example 3

The following N. meningitidis DNA sequence was identified <SEQ ID 13>: 1 ATGAAACTTC TGACCACCGC AATCCTGTCT TCCGCAATCG CGCTCAGCAG 51 TATGGCTGCC GCCGCTGGCA CGGACAACCC CACTGTTGCA AAAAAAACCG 101 TCAGCTACGT CTGCCAGCAA GGTAAAAAAG TCAAAGTAAC CTACGGCTTC 151 AACAAACAGG GTCTGACCAC ATACGCTTCC GCCGTCATCA ACGGCAAACG 201 CGTGCAAATG CCTGTCAATT TGGACAAATC CGACAATGTG GAAACATTCT 251 ACGGCAAAGA AGGCGGTTAT GTTTTGGGTA CCGGCGTGAT GGATGGCAAA 301 TCCTACCGCA AACAGCCCAT TATGATTACC GCACCTGACA ACCAAATCGT 351 CTTCAAAGAC TGTTCCCCAC GTTAA

This corresponds to the amino acid sequence <SEQ ID 14; ORF44>: 1 MKLLTTAILS SAIALSSMAA AAGTDWPTVA KKTVSYVCQQ GKKVKVTYGF 51 NKQGLTTYAS AVINGKRVQH PVNLDKSDNV ETFYGKEGGY VLGTGVMDGK 101 SYRKQPIHIT APDNQIVFKD CSPR*

Computer analysis of this amino acid sequence predicted the leader peptide shown underlined.

Further work identified the corresponding gene in strain A of N. meningitidis <SEQ ID 15>: 1 ATGAAACTTC TGACCACCGC AATCCTGTCT TCCGCAATCG CGCTCAGCAG 51 TATGGCTGCT GCTGCCGGCA CGAACAACCC CACCGTTGCC AAAAAAACCG 101 TCAGCTACGT CTGCCAGCAA GGTAAAAAAG TCAAAGTAAC CTACGGCTTT 151 AACAAACAGG GCCTGACCAC ATACGCTTCC GCCGTCATCA ACGGCAAACG 201 TGTGCAAATG CCTGTCAATT TGGACAAATC CGACAATGTG GAAACATTCT 251 ACGGCAAAGA AGGCGGTTAT GTTTTGGGTA CCGGCGTGAT GGATGGCAAA 301 TCCTATCGCA AACAGCCTAT TATGATTACC GCACCTGACA ACCAAATCGT 351 CTTCAAAGAC TGTTCCCCAC GTTAA

This encodes a protein having amino acid sequence <SEQ ID 16; ORF44a>: 1 MKLLTTAILS SAIALSSMAA AAGTNNPTVA KKTVSYVCQQ GKKVKVTYGF 51 NKQGLTTYAS AVINGKRVQM PVNLDKSDNV ETFYGKEGGY VLGTGVMDGK 101 SYRKQPIMIT APDNQIVFKD CSPR*

The strain B sequence (ORF44) shows 99.2% identity over a 124 aa overlap with ORF44a:         10        20        30        40        50        60 orf44.pep MKLLTTAILSSAIALSSMAAAAGTDNPTVAKKTVSYVCQQGKKVKVTYGFNKQGLTTYAS ||||||||||||||||||||||||:||||||||||||||||||||||||||||||||||| orf44a MKLLTTAILSSAIALSSMAAAAGTNNPTVAKKTVSYVCQQGKKVKVTYGFNKQGLTTYAS         10        20        30        40        50        60         70        80        90       100       110       120 orf44.pep AVINGKRVQMPVNLDKSDNVETFYGKEGGYVLGTGVMDGKSYRKQPIMITAPDNQIVFKD |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| orf44a AVINGKRVQMPVNLDKSDNVETFYGKEGGYVLGTGVMDGKSYRKQPIMITAPDNQIVFKD         70        80        90       100       110       120 orf44.pep CSPRX ||||| orf44a CSPRX

Computer analysis gave the following results:

Homology with the LecA Adhesin of Eikenella corrodens (Accession Number D78153)

ORF44 and LecA protein show 45% aa identity in 91 aa overlap: Orf44  33 TVSYVCQQGKKVKVTYGFNKQGLTTYASAVINGKRVQMPVNLDKSDNVETFYGKEGGYVL 92     +V+YVCQQG+++ V Y FN  G+ T A   +N + +++P NL  SDNV+T +    GY L LecA 135 SVAYVCQQGRRLNVNYRFNSAGVPTSAELRVNNRNLRLPYNLSASDNVDTVF-SANGYRL 193 Orf44  93 GTGVHDGKSYRKQPIHITAPDNQIVFKDCSP 123      T  MD  +YR Q I+++AP+ Q+++KDCSP LecA 194 TTNAMDSANYRSQDIIVSAPNGQNLYKDCSP 224

Based on homology with the adhesin, it was predicted that this protein from N. meningitidis, and its epitopes, could be useful antigens for vaccines or diagnostics.

ORF44-1 (11.2 kDa) was cloned in pET and pGex vectors and expressed in E. coli, as described above. The products of protein expression and purification were analyzed by SDS-PAGE. FIG. 3A shows the results of affinity purification of the His-fusion protein, and FIG. 3B shows the results of expression of the GST-fusion in E-coli. Purified His-fusion protein was used to immunise mice, whose sera were used for ELISA, which gave positive results, and for a bactericidal assay (FIG. 3C). These experiments confirm that ORF44-1 is a surface-exposed protein, and that it is a useful immunogen.

FIG. 3D shows plots of hydrophilicity, antigenic index, and AMPHI regions for ORF44-1.

Example 4

The following partial DNA sequence was identified in N. meningitidis <SEQ ID 17> 1 GGCACCGAAT TCAAAACCAC CCTTTCCGGA GCCGACATAC AGGCAGGGGT 51 GGGTGAAAAA GCCCGAGCCG ATGCGAAAAT TATCCTAAAA GGCATCGTTA 101 ACCGCATCCA AACCGAAGAA AAGCTGGAAT CCAACTCGAC CGTATGGCAA 151 AAGCAGGCCG GAAGCGGCAG CACGGTTGAA ACGCTGAAGC TACCGAGCTT 201 TGAAGGGCCG GCACTGCCTA AGCTGACCGC TCCCGGCGGC TATATCGCCG 251 ACATCCCCAA AGGCAACCTC AAAACCGAAA TCGAAAAGCT GGCCAAACAG 301 CCCGAATATG CCTATCTGAA ACAGCTTCAG ACGGTCAAGG ACGTGAACTG 351 GAACCAAGTA CAGCTCGCTT ACGACAAATG GGACTATAAA CAGGAAGGCC 401 TAACCGGAGC CGGAGCCGCA ATTANCGCAC TGGCCGTTAC CGTGGTCACC 451 TCAGGCGCAG GAACCGGAGC CGTATTGGGA TTAANACGNG TGGCCGCCGC 501 CGCAACCGAT GCAGCATTT...

This corresponds to the amino acid sequence <SEQ ID 18; ORF49>: 1 GTEFKTTLSG ADIQAGVGEK ARADPKIILK GIVNRIQTEE KLESNSTVWQ 51 KQAGSGSTVE TLKLPSFEGP ALPKLTAPGG YIADIPKGNL KTEIEKLAKQ 101 PEYAYLKQLQ TVKDVNWNQV QLAYDKWDYK QEGLTCAGAA IXALAVTVVT 151 SGAGTGAVLG LXRVAAAATD AAF..

Further work revealed the complete nucleotide sequence <SEQ ID 19>: 1 ATGCAACTGC TGGCAGCCGA AGGCATTCAC CAACACCAAT TGAATGTTCA 51 GAAAAGTACC CGTTTCATCG GCATCAAAGT GGGTAAAAGC AATTACAGCA 101 AAAACGAGCT GAACGAAACC AAACTGCCCG TACGCGTTAT CGCCCAAACA 151 GCCAAAACCC GTTCCGGCTG GGATACCGTA CTCGAAGGCA CCGAATTCAA 201 AACCACCCTT TCCGGAGCCG ACATACAGGC AGGGGTGGGT GAAAAAGCCC 251 GAGCCGATGC GAAAATTATC CTAAAAGGCA TCGTTAACCG CATCCAAACC 301 GAAGAAAAGC TGGAATCCAA CTCGACCGTA TGGCAAAAGC AGGCCGGAAG 351 CGGCAGCACG GTTGAAACGC TGAAGCTACC GAGCTTTGAA GGGCCGGCAC 401 TGCCTAAGCT GACCGCTCCC GGCGGCTATA TCGCCGACAT CCCCAAAGGC 451 AACCTCAAAA CCGAAATCGA AAAGCTGGCC AAACAGCCCG AATATGCCTA 501 TCTGAAACAG CTTCAGACGG TCAAGGACGT GAACTGGAAC CAAGTACAGC 551 TCGCTTACGA CAAATGGGAC TATAAACAGG AAGGCCTAAC CGGAGCCGGA 601 GCCGCAATTA TCGCACTGGC CGTTACCGTG GTCACCTCAG GCGCAGGAAC 651 CGGAGCCGTA TTGGGATTAA ACGGTGCGGC CGCCGCCGCA ACCGATGCAG 701 CATTTGCCTC TTTGGCCAGC CAGGCTTCCG TATCGTFCAT CAACAACAAA 751 CGCAATATCG GTAACACCCT GAAAGAGCTG GGCAGAAGCA GCACGGTGAA 801 AAATCTGATG GTTGCCGTCG CTACCGCAGG CGTAGCCGAC AAAATCGGTG 851 CTTCGGCACT GAACAATGTC AGCGATAAGC AGTGGATCAA CAACCTGACC 901 GTCAACCTGG CCAATGCGGG CAGTGCCGCA CTGATTAATA CCGCTGTCAA 951 CGGCGGCAGC CTGAAAGACA ATCTGGAAGC GAATATCCTT GCGGCTTTGG 1001 TGAATACTGC GCATGGAGAG GCAGCAAGTA AAATCAAACA GTTGGATCAG 1051 CACTACATTG CCCATAAGAT TGCCCATGCC ATAGCGGGCT GTGCGGCAGC 1101 GGCGGCGAAT AAGGGCAAGT GTCAAGATGG TGCGATCGGT GCGGCGGTCG 1151 GTGAAATCCT TGGCGAAACC CTACTGGACG GCAGAGACCC TGGCAGCCTG 1201 AATGTGAAGG ACAGGGCAAA AATCATTGCT AAGGCGAAGC TGGCAGCAGG 1251 GGCGGTTGCG GCGTTGAGTA AGGGGGATGT GAGTACGGCG GCGAATGCGG 1301 CTGCTGTGGC GGTAGAGAAT AATTCTTTAA ATOATATACA GGATCGTTTG 1351 TTGAGTGGAA ATTATGCTTT ATGTATGAGT GCAGGAGGAG CAGAAAGCTT 1401 TTGTGAGTCT TATCGACCAC TGGGCTTGCC ACACTTTGTA AGTGTTTCAG 1451 GAGAAATGAA ATTACCTAAT AAATTCGGGA ATCGTATGGT TAATGGAAAA 1531 TTAATTATTA ACACTAGAAA TGGCAATGTA TATTTCTCTG TAGGTAAAAT 1551 ATGGAGTACT GTAAAATCAA CAAAATCAAA TATAAGTGGG GTATCTGTCG 1601 GTTGGGTTTT AAATGTTTCC CCTAATGATT ATTTAAAAGA AGCATTTATG 1651 AATGATTTCA GAAATAGTAA TCAAAATAAA GCCTATGCAG AAATGATTTC 1701 CCAGACTTTG GTAGGTGAGA GTGTTGGTGG TAGTCTTTGT CTGACAAGAG 1751 CCTGCTTTTC GGTAAGTTCA ACAATATCTA AATCTAAATC TCCTTTTAAA 1801 GATTCAAAAA TTATTGGGGA AATCGGTTTG GGAAGTGGTG TTGCTGCAGG 1851 AGTAGAAAAA ACAATATACA TAGGTAACAT AAAAGATATT GATAAATTTA 1901 TTAGTGCAAA CATAAAAAAA TAG

This corresponds to the amino acid sequence <SEQ ID 20; ORF49-1>: 1 MQLLAAEGIH QHQLNVQKST RFIGIKVGKS NYSKNELNET KLPVRVIAQT 51 AKTRSGWDTV LEGTEFKTTL SGADIQAGVG EKARADAKII LKGIVNRIQT 101 EEKLESNSTV WQKQAGSGST VETLKLPSFE GPALPKLTAP GGYIADIPKG 151 NLKTEIEKLA KQPEYAYLKQ LQTVKDVNWN QVQLAYDKWD YKQEGLTGAG 201 AAIIALAVTV VTSGAGTGAV LGLNGAAAAA TDAAFASLAS QASVSFINNK 251 GNIGNTLKEL GRSSTVKNLM VAVATAGVAD KIGASALNNV SDKQWINNLT 301 VNLANAGSAA LINTAVNGGS LKDNLEANIL AALVNTAHGE AASKIKQLDQ 351 HYIAHKIAHA IAGCAAAAAN KGKCQDGAIG AAVGEILGET LLDGRDPGSL 401 NVKDRAKIIA KAKLAAGAVA ALSKGDVSTA ANAAAVAVEN NSLNDIQDRL 451 LSGNYALCNS AGGAESFCES YRPLGLPHFV SVSGENKLPN KFGNRNVNGK 501 LIINTRNGNV YFSVGKIWST VKSTKSNISG VSVGWVLNVS PNDYLKEASM 551 NDFRNSNQNK AYAEMISQTL VGESVGGSLC LTRACFSVSS TISKSKSPFK 601 DSKIIGEIGL GSGVAAGVEK TIYIGNIKDI DKFISANIKK *

Computer analysis predicts a transmembrane domain and also indicates that ORF49 has no significant amino acid homology with known proteins. A corresponding ORF from N. meningitidis strain A was, however, identified:

ORF49 shows 86.1% identity over a 173 aa overlap with an ORF (ORF49a) from strain A of N. meningitidis:                                        10        20        30 orf49.pep                                GTEFKTTLSGADIQAGVGEKARADAKIILK                                ||||||||:|||||||| ||||:||||||| orf49a SKNELNETKLPVRVVAQXAATRSGWDTVLEGTEFKTTLAGADIQAGVXEKARVDAKIILK       40        50        60        70        80        90         40        50        60        70        80        90 orf49.pep GIVNRIQTEEKLESNSTVWQKQAGSGSTVETLKLPSFEGPALPKLTAPGGYIADIPKGNL |||||||:|||||:|||||||||| |||:|||||||||:|: |||:||||||:||||||| orf49a GIVNRIQSEEKLETNSTVWQKQAGRGSTIETLKLPSFESPTPPKLSAPGGYIVDIPKGNL      100       110       120       130       140       150        100       110       120       130       140       150 orf49.pep KTEIEKLAKQPEYAYLKQLQTVKDVNWNQVQLAYDKWDYKQEGLTGAGAAIXALAVTVVT |||||||:||||||||||||::|::||||||||||:||||||||| ||||| |||||||| orf49a KTEIEKLSKQPEYAYLKQLQVAKNINWNQVQLAYDRWDYKQEGLTEAGAAIIALAVTVVT      160       170       180       190       200       210        160       170 orf49.pep SGAGTGAVLGLXRVAAAATDAAF |||||||||||  : |||||||| orf49a SGAGTGAVLGLNGAXAAATDAAFASLASQASVSFINNKGDVGKTLKELGRSSTVKNLVVA      220       230       240       250       260       270

ORF49-1 and ORF49a show 83.2% identity in 457 aa overlap: orf49a.pep XQLLAEEGIHKHELDVQKSRRFIGIKVGXSNYSKNELNETKLPVRVVAQXAATRSGWDTV  |||| ||||:|:|:|||| |||||||| |||||||||||||||||:||:| |||||||| orf49-1 MQLLAAEGIHQHQLNVQKSTRFIGIKVGKSNYSKNELNETKLPVRVIAQTAKTRSGWDTV orf49a.pep LEGTEFKTTLAGADIQAGVXEKARVDAKIILKGIVNRIQSEEKLETNSTVWQKQAGRGST ||||||||||:|||||||| ||||:||||||||||||||:|||||:|||||||||| ||| orf49-1 LEGTEFKTTLSGADIQAGVGEKARADAXIILKGIVNRIQTEEKLESNSTVWQKQAGSGST orf49a.pep IETLKLPSFESPTPPKLSAPGGYIVDIPKGNLKTEIEKLSKQPEYAYLKQLQVAKNINWN :|||||||||:|: |||:||||||:||||||||||||||:||||||||||||::|::||| orf49-1 VETLKLPSFEGPALPKLTAPGGYIADIPKGNLKTEIEKLAKQPEYAYLKQLQTVKDVNWN orf49a.pep QVQLAYDRWDYKQEGLTEAGAAIIALAVTVVTSGAGTGAVLGLNGAXAAATDAAFASLAS |||||||:||||||||| |||||||||||||||||||||||||||| ||||||||||||| orf49-1 QVQLAYDKWDYKQEGLTGAGAAIIALAVTVVTSGAGTGAVLGLNGAAAAATDAAFASLAS orf49a.pep QASVSFINNKGDVGKTLKELGRSSTVKNLVVAAATAGVADKIGASALXNVSDKQWINNLT |||||||||||::|:||||||||||||||:||:|||||||||||||| |||||||||||| orf49-1 QASVSFINNKGNIGNTLKELGRSSTVKNLMVAVATAGVADKIGASALNNVSDKQWINNLT orf49a.pep VNLANAGSAALINTAVNGGSLKDXLEANILAALVNTAHGEAASKIKQLDQHYIVHKIAHA ||||||||||||||||||||||| |||||||||||||||||||||||||||||:|||||| orf49-1 VNLANAGSAALINTAVNGGSLKDNLEANILAALVNTAHGEAASKIKQLDQHYIAHKIAHA orf49a.pep IAGCAAAAANKGKCQDGAIGAAVGEIVGEALTNGKNPDTLTAKEREQILAYSKLVAGTVS ||||||||||||||||||||||||||:||:| :|::| :|::|:| :|:| :||:||:|: orf49-1 IAGCAAAAANKGKCQDGAIGAAVGEILGETLLDGRDPGSLNVKDRAKIIAKAKLAAGAVA orf49a.pep GVVGGDVNAAANAAEVAVKNNQLSDXEGREFDNEMTACAKQNXPQLCRKNTVKKYQNVAD ::  |||::||||| |||:||:|:| : | :::::: | orf49-1 ALSKGDVSTAANAAAVAVENNSLNDIQDRLLSGNYALCMSAGGAESFCESYRPLGLPHFV orf49a.pep KRLAASIAICTDISRSTECRTIRKQHLIDSRSLHSSWEAGLIGKDDEWYKLFSKSYTQAD orf49-1 SVSGEMKLPNKFGNRMVNGKLIINTRNGNVYFSVGKIWSTVKSTKSNISGVSVGWVLNVS

The complete length ORF49a nucleotide sequence <SEQ ID 21> is: 1 NTGCAACTGC TGGCAGAAGA AGGCATCCAC AAGCACGAGT TGGATGTCCA 51 AAAAAGCCGC CGCTTTATCG GCATCAAGGT AGGTNAGAGC AATTACAGTA 101 AAAACGAACT GAACGAAACC AAATTGCCTG TCCGCGTCGT CGCCCAAANT 151 GCAGCCACCC GTTCAGGCTG GGATACCGTG CTCGAAGGTA CCGAATTCAA 201 AACCACGCTG GCCGGTGCCG ACATTCAGGC AGGTGTANGC GAAAAAGCCC 251 GTGTCGATGC GAAAATTATC CTCAAAGGCA TTGTGAACCG TATCCAGTCG 301 GAAGAAAAAT TAGAAACCAA CTCAACCGTA TGGCAGAAAC AGGCCGGACG 351 CGGCAGCACT ATCGAAACGC TAAAACTGCC CAGCTTCGAA AGCCCTACTC 401 CGCCCAAATT GTCCGCACCC GGCGGNTATA TCGTCGACAT TCCGAAAGGC 451 AATCTGAAAA CCGAAATCGA AAAGCTGTCC AAACAGCCCG AGTATGCCTA 501 TCTGAAACAG CTCCAAGTAG CGAAAAACAT CAACTGGAAT CAGGTGCAGC 551 TTGCTTACGA CAGATGGGAC TACAAACAGG AGGGCTTAAC CGAAGCAGGT 601 GCGGCGATTA TCGCACTGGC CGTTACCGTG GTCACCTCAG GCGCAGGAAC 651 CGGAGCCGTA TTGGGATTAA ACGGTGCGNC CGCCGCCGCA ACCGATGCAG 701 CATTCGCCTC TTTGGCCAGC CAGGCTTCCG TATCGTTCAT CAACAACAAA 751 GGCGATGTCG GCAAAACCCT GAAAGAGCTG GGCAGAAGCA GCACGGTGAA 801 AAATCTGGTG GTTGCCGCCG CTACCGCAGG CGTAGCCGAC AAAATCGGCG 851 CTTCGGCACT GANCAATGTC AGCGATAAGC AGTGGATCAA CAACCTGACC 901 GTCAACCTAG CCAATGCGGG CAGTGCCGCA CTGATTAATA CCGCTGTCAA 951 CGGCGGCAGC CTGAAAGACA NTCTGGAAGC GAATATCCTT GCGGCTTTGG 1001 TCAATACCGC GCATGGAGAA GCAGCCAGTA AAATCAAACA GTTGGATCAG 1051 CACTACATAG TCCACAAGAT TGCCCATGCC ATAGCGGGCT GTGCGGCAGC 1101 GGCGGCGAAT AAGGGCAAGT GTCAGGATGG TGCGATAGGT GCGGCTGTGG 1151 GCGAGATAGT CGGGGAGGCT TTGACAAACG GCAAAAATCC TGACACTTTG 1201 ACAGCTAAAG AACGCGAACA GATTTTGGCA TACAGCAAAC TGGTTGCCGG 1251 TACGGTAAGC GGTGTGGTCG GCGGCGATGT AAATGCGGCG GCGAATGCGG 1301 CTGAGGTAGC GGTGAAAAAT AATCAGCTTA GCGACTAAGA GGGTAGAGAA 1351 TTTGATAACG AAATGACTGC ATGCGCCAAA CAGAATANTC CTCAACTGTG 1401 CAGAAAAAAT ACTGTAAAAA AGTATCAAAA TGTTGCTGAT AAAAGACTTG 1451 CTGCTTCGAT TGCAATATGT ACGGATATAT CCCGTAGTAC TGAATGTAGA 1501 ACAATCAGAA AACAACATTT GATCGATAGT AGAAGCCTTC ATTCATCTTG 1551 GGAAGCAGGT CTAATTGGTA AAGATGATGA ATGGTATAAA TTATTCAGCA 1601 AATCTTACAC CCAAGCAGAT TTGGCTTTAC AGTCTTATCA TTTGAATACT 1651 GCTGCTAAAT CTTGGCTTCA ATCGGGCAAT ACAAAGCCTT TATCCGAATG 1701 GATGTCCGAC CAAGGTTATA CACTTATTTC AGGAGTTAAT CCTAGATTCA 1751 TTCCAATACC AAGAGGGITF GTAAAACAAA ATACACCTAT TACTAATGTC 1801 AAATACCCGG AAGGCATCAG TTTCGATACA AACCTANAAA GACATCTGGC 1851 AAATGCTGAT GGTTTTAGTC AAGAACAGGG CATTAAAGGA GCCCATAACC 1901 GCACCAATNT TATGGCAGAA CTAAATTCAC GAGGAGGANG NGTAAAATCT 1951 GAAACCCANA CTGATATTGA AGGCATTACC CGAATTAAAT ATGAGATTCC 2001 TACACTAGAC AGGACAGGTA AACCTGATGG TGGATTTAAG GAAATTTCAA 2051 GTATAAAAAC TGTTTATAAT CCTAAAAANT TTTNNGATGA TAAAATACTT 2101 CAAATGGCTC AANATGCTGN TTCACAAGGA TATTCAAAAG CCTCTAAAAT 2151 TGCTCAAAAT GAAAGAACTA AATCAATATC GGAAAGAAAA AATGTCATTC 2201 AATTCTCAGA AACCTTTGAC GGAATCAAAT TTAGANNNTA TNTNGATGTA 2251 AATACAGGAA GAATTACAAA CATTCACCCA GAATAATTTA A

This encodes a protein having amino acid sequence <SEQ ID 22>: 1 XQLLAEEGIH KHELDVQKSR RFIGIKVGXS NYSKNELNET KLPVRVVAQX 51 AATRSGWDTV LEGTEFKTTL AGADIQAGVX EKARVOAKII LKGIVNRIQS 101 EEKLETNSTV WQKQAGRGST IETLKLPSFE SPTPPKLSAP GGYIVDIPKG 151 NLKTEIEKLS KQPEYAYLKQ LQVAKNINWN QVQLAYDRWD YKQEGLTEAG 201 AAIIALAVTV VTSGAGTGAV LGLNGAXAAA TORAFASLAS QASVSFINNK 251 GDVGKTLKEL GRSSTVKNLV VAAATAGVAD KIGASALXNV SDKQNINNLT 301 VNLANAGSAA LINTAVNGGS LKDXLEANIL AALVNTAHGE AASKIKQLDQ 351 HYIVHKIAHA IAGCAAAAAN KGKCQDGAIG AAVGEIVGEA LTNGKNPDTL 401 TAKEREQILA YSKLVAGTVS GVVGGDVNAA ANAAEVAVKN NQLSDXEGRE 451 FDWEHTACAK QNXPQLCRXN TVKKYQNVAD KRLAASIAIC TDISRSTECR 501 TIRKQHLIDS RSLHSSWEAG LIGKDDEWYK LFSKSYTQAD LALOSYHLNT 551 AAKSWLQSGN TKPLSEWNSD QGYTLISGVN PRFIPIPRGF VKQNTPITNV 601 KYPEGISFDT NLXRHLATAD GFSQEQGIKG AHNRTNXMAE LNSRGGXVKS 651 ETXTDIEGIT RIKYEIPTLD RTGKPDGGFK EISSIKTVYN FKXFKDDKIL 701 QMAQXAXSQG YSKASKIAQN ERTKSISERK NVIQFSETFD GIKFRXYXDV 751 NTGRITNIHP E

Based on the presence of a putative transmembrane domain, it is predicted that these proteins from N. meningitidis, and their epitopes, could be useful antigens for vaccines or diagnostics.

Example 5

The following partial DNA sequence was identified in N. meningitidis SEQ ID 23> 1 ..CGGATCGTTG TAGGTTTGCG GATTTCTTGC GCCGTAGTCA CCGTAGTCCC 51   AAGTATAACC CAAGGCTTTG TCTTCGCCTT TCATTCCGAT AAGGGATATG 101   ACGCTTTGGT CGGTATAGCC GTCTTGGGAA CCTTTGTCCA CCCAACGCAT 151   ATCTGCCTGC GGATTCTCAT TGCCGCTTCT TGGCTGCTGA TTTTTCTGCC 201   TTCGCGTTTT TCAACTTCGC GCTTGAGGGC TTCGGCATAT TTGTCGGCCA 251   ACGCCATTTC TTTCGGATGC AGCTGCCTAT TGTTCCAATC TACATTCGCA 301   CCCACCACAG CACCACCACT ACCACCAGTT GCATAG

This corresponds to the amino acid sequence <SEQ ID 24; ORF50>: 1 ..RIVVGLRISC AVVTVVPSIT QGFVFAFHSD KGYDALVGIA VLGTFVHPTH 51 ICLRILIAAS WLLIFLPSRF STSRLRASAY LSANAISFGC SCLLFQSTFA 101 PTTAPPLPPV A*

Computer analysis predicts two transmembrane domains and also indicates that ORF50 has no significant amino acid homology with known proteins.

Based on the presence of a putative transmembrane domain, it is predicted that this protein from N. meningitidis, and its epitopes, could be useful antigens for vaccines or diagnostics.

Example 6

The following partial DNA sequence was identified in N. meningitidis <SEQ ID 25> 1 ..AAGTTTGACT TTACCTGGTT TATTCCGGCG GTAATCAAAT ACCGCCGGTT 51   GTTTTTTGAA GTATTGGTGG TGTCGGTGGT GTTGCAGCTG TTTGCGCTGA 101   TTACGCCTCT GTTTTTCCAA GTGGTGATGG ACAAGGTGCT GGTACATCGG 151   GGATTCTCTA CTTTGGATGT GGTGTCGGTG GCTTTGTTGG TGGTGTCGCT 201   GTTTGAGATT GTGTTGGGCG GTTTGCGGAC GTATCTGTTT GCACATACGA 251   CTTCACGTAT TGATGTGGAA TTGGGCGCGC GTTTGTTCCG GCATCTGCTT 301   TCCCTGCCTT TATCCTATTT CGAGCACAGA CGAGTGGGTG ATACGGTGGC 351   TCGGGTGCGG GAATTGGAGC AGATTCGCAA TTTCTTGACC GGTCAGGCGC 401   TGACTTCGGT GTTGGATTTG GCGTTTTCGT TTATCTTTCT GGCGGTGATG 451   TGGTATTACA GCTCCACTCT GACTTGGGTG GTATTGGCTT CGTTG.....                               // 1451   .......... .......... .......... .......... .......... 1501   .......... .......... .......... .......... ..ATTTGCGC 1551   CAACCGGACG GTGCTGATTA TCGCCCACCG TCTGTCCACT GTTAAAACGG 1601   CACACCGGAT CATTGCCATG GATAAAGGCA GGATTGTGGA AGCGGGAACA 1651   CAGCAGGAAT TGCTGGCGAA CG..AACGGA TATTACCGCT ATCTGTATGA 1701   TTTACAGAAC GGGTAG

This corresponds to the amino acid sequence <SEQ ID 26; ORF39>: 1 ..KFDFTWFIPA VIKYRRLFFE VLVVSVVLQL FALITPLFFQ VVMDKVLVHR 51   GFSTLDVVSV ALLVVSLFEI VLGGLRTYLF AHTTSRIDVE LGARLFRHLL 101   SLPLSYFEHP RVGDTVARVR ELEQIRNFLT GQALTSVLDL AFSFIFLAVM 151   WYYSSTLTWV VLASL..... .......... .......... ..........                              // 501   .......... ....ICANRT VLIIAHRLST VKTAHRIIAH DKGRIVEAGT 551   QQELLANXNG YYRYLYDLQN G*

Further work revealed the complete nucleotide sequence <SEQ ID 27>: 1 ATGTCTATCG TATCCGCACC GCTCCCCGCC CTTTCCGCCC TCATCATCCT 51 CGCCCATTAC CACGGCATTG CCGCCAATCC TGCCGATATA CAGCATGAAT 101 TTTGTACTTC CGCACAGAGC GATTTAAATG AAACGCAATG GCTGTTAGCC 151 GCCAAATCTT TGGGATTGAA GGCAAAGGTA GTCCGCCAGC CTATTAAACG 201 TTTGGCTATG GCGACTTTAC CCGCATTGGT ATGGTGTGAT GACGGCAACC 251 ATTTCATTTT GGCCAAAACA GACGGTGAGG GTGAGCATGC CCAATTTTFG 301 ATACAGGATT TGGTTACGAA TAAGTCTGCG GTATTGTCTT TTGCCGAATT 351 TTCTAACAGA TATTCGGGCA AACTGATATT GGTTGCTTCC CGCGCTTCGG 401 TATTGGGCAG TTTGGCAAAG TTTGACTTTA CCTGGTTTAT TCCGGCGGTA 451 ATCAAATACC GCCGGTTGTT TTTTGAAGTA TTGGTGGTGT CGGTGGTGTT 501 GCAGCTGTTT GCGCTGATTA CGCCTCTGTT TTTCCAAGTG GTGATGGACA 551 AGGTGCTGGT ACATCGGGGA TTCTCTACTT TGGATGTGGT GTCGGTGGCT 601 TTGTTGGTGG TGTCGCTGTT TGAGATTGTG TTGGGCGGTT TGCGGACGTA 651 TCTGTTTGCA CATACGACTT CACGTATTGA TGTGGAATTG GGCGCGCGTT 701 TGTTCCGGCA TCTGCTTTCC CTGCCTTTAT CCTATTTCGA GCACAGACGA 751 GTGGGTGATA CGGTGGCTCG GGTGCGGGAA TTGGAGCAGA TTCGCAATTT 801 CTTGACCGGT CAGGCGCTGA CTTCGGTGTT GGATTTGGCG TTTTCGTTTA 951 TCTTTCTGGC GGTGATGTGG TATTACAGCT CCACTCTGAC TTGGGTGGTA 901 TTGGCTTCGT TGCCTGCCTA TGCGTTTTGG TCGGCATTTA TCAGTCCGAT 951 ACTGCGGACG CGTCTGAACG ATAAGTTCGC GCGCAATGCA GACAACCAGT 1001 CGTTTTTAGT AGAAAGCATC ACTGCGGTGG GTACGGTAAA GGCGATGGCG 1051 GTGGAGCCGC AGATGACGCA GCGTTGGGAC AATCAGTTGG CGGCTTATGT 1101 GGCTTCGGGA TTTCGGGTAA CGAAGTTGGC GGTGGTCGGC CAGCAGGGGG 1151 TGCAGCTGAT TCAGAAGCTG GTGACGGTGG CGACGTTGTG GATTGGCGCA 1201 CGGCTGGTAA TTGAGAGCAA GCTGACGGTG GGGCAGCTGA TTGCGTTTAA 1251 TATGCTCTCG GGACACGTGG CGGCGCCTGT TATCCGTTTG GCGCAGTTGT 1301 GGCAGGATTT CCAGCAGGTG GGGATTTCGG TGGCGCGTTT GGGGGATATT 1351 CTGAATGCGC CGACCGAGAA TGCGTCTTCG CATTTGGCTT TGCCCGATAT 1401 CCGGGGGGAG ATTACGTTCG AACATGTCGA TTTCCGCTAT AAGGCGGACG 1451 GCAGGCTGAT TTTGCAGGAT TTGAACCTGC GGATTCCGGC GGGGGAAGTG 1501 CTGGGGATTG TGGGACGTTC GGGGTCGGGC AAATCCACAC TCACCAAATT 1551 GGTGCAGCGT CTGTATGTAC CGGAGCAGGG ACGGGTGTTG GTGGACGGCA 1601 ACGATTTGGC TTTGGCCGCT CCTGCCTGGC TGCGGCGGCA GGTCGGCGTG 1651 GTCTTGCAGG AGAATGTGCT GCTCAACCGC AGCATACGCG ACAATATCGC 1701 GCTGACGGAT ACGGGTATGC CGCTGGAACG CATTATCGAA GCAGCCAAAC 1751 TGGCGGGCGC ACACGAGTTT ATTATGGAGC TGCCGGAAGG CTACGGCACC 1801 GTGGTGGGCG AACAAGGGGC CGGCTTGTCG GGCGGACAGC GGCAGCGTAT 1951 TGCGATTGCC CGCGCGTTAA TCACCAATCC GCGCATTCTG ATTTTTGATG 1901 AAGCCACCAG CGCGCTGGAT TATGAAAGTG AACGAGCGAT TATGCAGAAC 1951 ATGCAGGCCA TTTGCGCCAA CCGGACGGTG CTGATTATCG CCCACCGTCT 2001 GTCCACTGTT AAAACGGCAC ACCGGATCAT TGCCATGGAT AAAGGCAGGA 2051 TTGTGGAAGC GGGAACACAG CAGGAATTGC TGGCGAAGCC GAACGGATAT 2101 TACCGCTATC TGTATGATTT ACAGAACGGG TAG

This corresponds to the amino acid sequence <SEQ ID 28; ORF39-1>: 1 MSIVSAPLPA LSALIILAMY HGIAANPADI QHEFCTSAQS DLNETQWLLA 51 AKSLGLKAKV VRQPIKRLAM ATLPALVWCD DGNHFILAKT DGEGEHAQFL 101 IQDLVTNKSA VLSFAEFSNR YSGKLILVAS RASVLGSLAK FDFTWFIPAV 151 IKYRRLFFEV LVVSVVLQLF ALITPLFFQV VMDKVLVHRG FSTLDVVSVA 201 LLVVSLFEIV LGGLRTYLFA HTTSRIDVEL GARLFRHLLS LPLSYFEHPA 251 VGDTVARVRE LEQIRNFLTG QALTSVLDLA FSFIFLAVMW YYSSTLTWVV 301 LASLPAYAFW SAFISPILRT RLNDKFAPNA DNQSFLVESI TAVGTVKAMA 351 VEPQHTQRWD NQLAAYVASG FRVTKLAVVG QQGVQLIQKL VTVATLWIGA 401 RLVIESRLTV GQLIAFNMLS GQVAAPVIRL AQLWQDFQQV GISVARLGDI 451 VVAPTENASS HLALPDIRGE ITFEHVDFRY KADGRLILQD LNLRIRAGEV 501 LGIVGRSGSG KSTLTKLVQR LYVPEQGRVL VDGNDLALAA PAWLRRQVGV 551 VLOENVLLNR SIRDNIALTD TGNPLERIIE AAKLAGAHEF IMELPEGYGT 601 VVGEQGAGLS GGQRQRIAIA RALITNPRIL IFDEATSALD YESERAINQN 651 MQAICANRTV LIIAHRLSTV KTAHRIIAND KGRIVEAGTQ QELLAKPNGY 701 YRYLYDLQNG *

Computer analysis of this amino acid sequence gave the following results:

Homology with a Predicted ORF from N. meningitidis (Strain A)

ORF39 shows 100% identity over a 165 aa overlap with an ORF (ORF39a) from strain A of N. meningitidis:                                         10        20        30 orf39.pep                                 KFDFTWFIPAVIKYRRLFFEVLVVSVVLQL                                 |||||||||||||||||||||||||||||| orf39a   AVLSFAEFSNRYSGKLILVASRASVLGSLAKFDFTWFIPAVIKYRRLFFEVLVVSVVLQL 110       120       130       140       150       160           40        50        60        70        80        90 orf39.pep   FALITPLFFQVVMDKVLVHRGFSTLDVVSVALLVVSLFEIVLGGLRTYLFAHTTSRIDVE   |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| orf39a   FALITPLFFQVVMDKVLVHRGFSTLDVVSVALLVVSLFEIVLGGLRTYLFAHTTSRIDVE 170       180       190       200       210       220          100       110       120       130       140       150 orf39.pep   LGARLFRHLLSLPLSYFEHRRVGDTVARVRELEQIRNFLTGQALTSVLDLAFSFIFLAVM   |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| orf39a   LGARLFRHLLSLPLSYFEHRRVGDTVARVRELEQIRNFLTGQALTSVLDLAFSFIFLAVM 230       240       250       260       270       280          160       170       180       190       200       210 orf39.pep   WYYSSTLTWVVLASLXXXXXXXXXXXXXXXXXXXXXXXXXXXXICANRTVLIIAHRLSTV   ||||||||||||||| orf39a   WYYSSTLTWVVLASLPAYAFWSAFISPILRTRLNDKFARNADNQSFLVESITAVGTVKAM 290       300       310       320       330       340

ORF39-1 and ORF39a show 99.4% identity in 710 aa overlap: orf39-1.pep MSIVSAPLPALSALIILAHYHGIAANPADIQHEFCTSAQSDLNETQWLLAAKSLGLKAKV |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| orf39a MSIVSAPLPALSALIILAHYHGIAANPADIQHEFCTSAQSDLNETQWLLAAKSLGLKAKV orf39-1.pep VRQPIKRLAMATLPALVWCDDGNHFILAKTDGEGEHAQFLIQDLVTNKSAVLSFAEFSNR |||||||||||||||||||||||||||||||| |||||:|||||:||||||||||||||| orf39a VRQPIKRLAMATLPALVWCDDGNHFILAKTDGGGEHAQYLIQDLTTNKSAVLSFAEFSNR orf39-1.pep YSGKLILVASRASVLGSLAKFDFTWFIPAVIKYRRLFFEVLVVSVVLQLFALITPLFFQV |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| orf39a YSGKLILVASRASVLGSLAKFDFTWFIPAVIKYRRLFFEVLVVSVVLQLFALITPLFFQV orf39-1.pep VMDKVLVHRGFSTLDVVSVALLVVSLFEIVLGGLRTYLFAHTTSRIDVELGARLFRHLLS |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| orf39a VMDKVLVHRGFSTLDVVSVALLVVSLFEIVLGGLRTYLFAHTTSRIDVELGARLFRHLLS orf39-1.pep LPLSYFEHRRVGDTVARVRELEQIRNFLTGQALTSVLDLAFSFIFLAVMWYYSSTLTWVV |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| orf39a LPLSYFEHRRVGDTVARVRELEQIRNFLTGQALTSVLDLAFSFIFLAVMWYYSSTLTWVV orf39-1.pep LASLPAYAFWSAFISPILRTRLNDKFARNADNQSFLVESITAVGTVKAMAVEPQMTQRWD |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| orf39a LASLPAYAFWSAFISPILRTRLNDKFARNADNQSFLVESITAVGTVKAMAVEPQMTQRWD orf39-1.pep NQLAAQVASGFRVTKLAVVGQQGVQLIQKLVTVATLWIGARLVIESKLTVGQLIAFNMLS |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| orf39a NQLAAQVASGFRVTKLAVVGQQGVQLIQKLVTVATLWIGARLVIESKLTVGQLIAFNMLS orf39-1.pep GQVAAPVIRLAQLWQDFQQVGISVARLGDILNAPTENASSHLALPDIRGEITFEHVDFRY |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| orf39a GQVAAPVIRLAQLWQDFQQVGISVARLGDILNAPTENASSHLALPDIRGEITFEHVDFRY orf39-1.pep KADGRLILQDLNLRIRAGEVLGIVGRSGSGKSTLTKLVQRLYVPEQGRVLVDGNDLALAA |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| orf39a KADGRLILQDLNLRIRAGEVLGIVGRSGSGKSTLTKLVQRLYVPEQGRVLVDGNDLALAA orf39-1.pep PAWLRRQVGVVLQENVLLNRSIRDNIALTDTGMPLERIIEAAKLAGAHEFIHELPEGYGT |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| orf39a PAWLRRQVGVVLQENVLLNRSIRDNIALTDTGMPLERIIEAAKLAGAHEFIHELPEGYGT orf39-1.pep VVGEQGAGLSGGQRQRIAIARALITNPRILIFDEATSALDYESERAIMQNMQAICANRTV |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| orf39a VVGEQGAGLSGGQRQRIAIARALITNPRILIFDEATSALDYESERAIMQNMQAICANRTV orf39-1.pep LIIAHRLSTVKTAHRIIAMDKGRIVEAGTQQELLAKPNGYYRYLYDLQNGX ||||||||||||||||||||||||||||||||||||||||||||||||||| orf39a LIIAHRLSTVKTAHRIIAMDKGRIVEAGTQQELLAKPNGYYRYLYDLQNGX

The complete length ORF39a nucleotide sequence <SEQ ID 29> is: 1 ATGTCTATCG TATCCGCACC GCTCCCCGCC CTTTCCGCCC TCATCATCCT 51 CGCCCATYAC CACGGCATTG CCGCCAATCC TGCCGATATA CAGCATGAAT 101 TTTGTACTTC CGCACAGAGC GATTTAAATG AAACGCAATG GCTGTTAGCC 151 GCCAAATCTT TGGGATTGAA GGCAAAGGTA GTCCGCCAGC CTATTAAACG 201 TTTGGCTATG GCGACTTTAC CCGCATTGGT ATGGTGTGAT GACGGCAACC 251 ATTTTATTTT GGCTAAAACA GACGGTGGGG GTGAGCATGC CCAATATCTA 301 ATACAGGATT TAACTACGAA TAAGTCTGCG GTATTGTCTT TTGCCGAATT 351 TTCTAACAGA TATTCGGGCA AACTGATATT GGTTGCTTCC CGCGCTTCGG 401 TATTGGGCAG TTTGGCAAAG TTTGACTTTA CCTGGTTTAT TCCGGCGGTA 451 ATCAAATACC GCCGGTTGTT TTTTGAAGTA TTGGTGGTGT CGGTGGTGTT 501 GCAGCTGTTT GCGCTGATTA CGCCTCTGTT TTTCCAAGTG GTGATGGACA 551 AGGTGCTGGT ACATCGGGGA TTCTCTATTT TGGATGTGGT GTCGGTGGCT 601 TTGTTGGTGG TGTCGCTGTT TGAGATTGTG TTGGGCGGTT TGCGGACGTA 651 TCTGTTTGCA CATACGACTT CACGTATTGA TGTGGAATTG GGCGCGCGTT 701 TGTTCCGGCA TCTGCTTTCC CTGCCTTTAT CCTATTTCGA GCACAGACGA 751 GTGGGTGATA CGGTGGCTCG GGTGCGGGAA TTGGAGCAGA TTCGCAATTT 801 CTTGACCGGT CAGGCGCTGA CTTCGGTGTT GGATTTGGCG TTTTCGTTTA 951 TCTTTCTGGC GGTGATGTGG TATTACAGCT CCACTCTGAC TTGGGTGGTA 901 TTGGCTTCGT TGCCTGCCTA TGCGTTTTGG TCGGCATTTA TCAGTCCGAT 951 ACTGCGGACG CGTCTGAACG ATAAGTTCGC GCGCAATGCA GACAACCAGT 1001 CGTTTTTAGT AGAAAGCATC ACTGCGGTGG GTACGGTAAA GGCGATGGCG 1051 GTGGAGCCGC AGATGACGCA GCGTTGGGAC AATCAGTTGG CGGCTTATGT 1101 GGCTTCGGGA TTTCGGGTAA CGAAGTTGGC GGTGGTCGGC CAGCAGGGGG 1151 TGCAGCTGAT TCAGAAGCTG GTGACGGTGG CGACGTTGTG GATTGGCGCA 1201 CGGCTGGTAA TTGAGAGCAA GCTGACGGTG GGGCAGCTGA TTGCGTTTAA 1251 TATGCTCTCG GGACAGGTGG CGGCGCCTGT TATCCGTTTG GCGCAGTTGT 1301 GGCAGGATTT CCAGCAGGTG GGGATTTCGG TGGCGCGTTT CGGGGATATT 1351 CTGAATGCGC CGACCGAGAA TGCGTCTTCG CATTTGGCTT TGCCCGATAT 1401 CCGGGGGGAG ATTACGTTCG AACATGTCGA TTTCCGCTAT AAGGCGGACG 1451 GCAGGCTGAT TTTGCAGGAT TTGAACCTGC GGATTCGGGC GGGGGAAGTG 1501 CTGGGGATTG TGGGACGTTC GGGGTCGGGC AAATCCACAC TCACCAAATT 1551 GGTGCAGCGT CTGTATGTAC CGGCGCAGGG ACGGGTGTTG GTGGACGGCA 1601 ACGATTTGGC TTTGGCCGCT CCTGCTTGGC TGCGGCGGCA GGTCGGCGTG 1651 GTCTTGCAGG AGAATGTGCT GCTCAACCGC AGCATACGCG ACAATATCGC 1701 GCTGACGGAT ACGGGTATGC CGCTGGAACG CATTATCGAA GCAGCCAAAC 1751 TGGCGGGCGC ACACGAGTTT ATTATGGAGC TGCCGGAAGG CTACGGCACC 1801 GTGGTGGGCG AACAAGGGGC CGGCTTGTCG GGCGGACAGC GGCAGCGTAT 1851 TGCGATTGCC CGCGCGTTAA TCACCAATCC GCGCATTCTG ATTTTTGATG 1901 AAGCCACCAG CGCGCTGGAT TATGAAAGTG AACGAGCGAT TATGCAGAAC 1951 ATGCAGGCCA TTTGCGCCAA CCGGACGGTG CTGATTATCG CCCACCGTCT 2001 GTCCACTGTT AAAACGGCAC ACCGGATCAT TGCCATGGAT AAAGGCAGGA 2051 TTGTGGAAGC GGGAACACAG CAGGAATTGC TGGCGAAGCC GAACGGATAT 2101 TACCGCTATC TGTATGATTT ACAGAACGGG TAG

This encodes a protein having amino acid sequence <SEQ ID 30>: 1 MSIVSAPLPA LSALIILAHY HGIAANPADI QHEFCTSAQS DLNETQWLLA 51 AKSLGLKAKV VRQPIKRLAN ATLPALVWCD DGNHFILAKT DGGGEHAQYL 101 IQDLTTNKSA VLSFAEFSNR YSGKLILVAS RASVIASLAX FDFTWFIPAV 151 IKYRRLFFEV LVVSVVLQLF ALITPLFFQV VNDKVLVHRG FSTLDVVSVA 201 LLVVSLFEIV LGGLRTYLFA HTTSRIDVEL GARLFRHLLS LPLSYFEHRR 251 VGDTVARVRE LEQIRNFLTG QALTSVLDLA FSFIFLAVMW YYSSTLTWVV 301 LASLPAYAFW SAFISPILRT RLNDKFARNA ONQSFLVESI TAVGTVKAMA 351 VEPQNTQRWD NQLAAYVASG FRVTKLAVVG QQGVQLIQKL VTVATLWIGA 401 RLVIESKLTV GQLIAFNHLS GQVAAPVIRL AQLWQOFQQV GISVARLGDI 451 LNAPTENASS HLALPDIRGE ITFEHVDFRY KADGRLILQD LNLRIRAGEV 501 LGIVGRSGSG KSTLTKLVQR LYVPAQGRVL VDGNDLALAA PAWLREQVGV 551 VLQENVLLNR SIRDNIALTD TGMPLERIIE AAKLAGAHEF IHPLPEGYGT 601 VVGEQGAGLS GGQRQRIAIA RALITNPRIL IFDEATSALD YESERAIMQN 651 NQAICANRTV LIIAHRLSTV KTAMRIIAMD KGRIVEAGTQ QELLAKPNGY 701 YRYLYOLQNG *

ORF39a is homologous to a cytolysin from A. pleuropneumoniae: sp|P26760|RT1B_ACTPL RTX-I TOXIN DETERMINANT B (TOXIN RTX-I SECRETION ATP- BINDING PROTEIN) (APX-IB) (HLY-IB) (CYTOLYSIN IB) (CLY-IB) >gi|97137|pir||D43599 cytolysin IB - Actinobacillus pleuropneumoniae (serotype 9) >gi|36944 (X61112) ClyI-B protein [Actinobacillus pleuropneumoniae] Length = 707 Score = 931 bits (2379), Expect =0.0 Identities = 472/690 (68%), Positives = 540/690 (77%), Gaps = 3/690 (0%) Query:  20 YHGIAANPADIQHEFCTSAQSDLNETQWXXXXXXXXXXXXVVRQPIKRLAMATLPALVWC 79            YH IA NP +++H+F    +  L+ T W             V++ I RLA   LPALVW Sbjct:  20 YHNIAVNPEELKHKFDLEGKG-LDLTAWLLAAKSLELKAKQVKKAIDRLAFIALPALVWR 78 Query:  80 DDGNHFILAKTDGGGEHAQYLIQDLTTNKSAVLSFAEFSNRYSGKLILVASRASVLGSLA 139            +DG HFIL K D   E  +YLI DL T+   +L  AEF + Y GKLILVASRAS++G LA Sbjct:  79 EDGKHFILTKIDN--EAKKYLIFDLETHNPRILEQAEFESLYQGKLILVASRASIVGKLA 136 Query: 140 KFDFTWFIPAVIKYRRXXXXXXXXXXXXXXXXXITPLFFQVVMDKVLVHRGFXXXXXXXX 199            KFDFTWFIPAVIKYR+                 ITPLFFQVVMDKVLVHRGF Sbjct: 137 KFDFTWFIPAVIKYRKIFIETLIVSIFLQIFALITPLFFQVVMDKVLVHRGFSTLNVITV 196 Query: 200 XXXXXXXFEIVLGGLRTYLFAHTTSRIDVELGARLFRHLLSLPLSYFEHRRVGDTVARVR 259                   FEIVL GLRTY+FAH+TSRIDVELGARLFRHLL+LP+SYFE+RRVGDTVARVR Sbjct: 197 ALAIVVLFEIVLNGLRTYIFAHSTSRIDVELGARLFRHLLALPISYFENRRVGDTVARVR 256 Query: 260 ELEQIRNFLTGQALTSVLDLAFSFIFLAVMWYYSSTLTWVVLASLPAYAFWSAFISPILR 319            EL+QIRNFLTGQALTSVLDL FSFIF AVMWYYS  LT V+L SLP Y  WS FISPILR Sbjct: 257 ELDQIRNFLTGQALTSVLDIMFSFIFFAVMWYYSPKLTLVILGSLPFYNGWSIFISPILR 316 Query: 320 TRLNDKFARNADNQSFLVESITAVGTVKAMAVEPQNTQRWDNQLAAYVASGFRVTKLAVV 379             RL++KFAR ADNQSFLVES+TA+ T+KA+AV PQMT  WD QLA+YV++GFRVT LA + Sbjct: 317 RRLDEKFARGADNQSFLVESVTAINTIKALAVTPQMTNTWDKQLASYVSAGFRVTTLATI 376 Query: 380 GQQGVQLIQKLVTVATLWIGARLVIESKLTVGOLIAFNNLSGQVAAPVIRLAQLWQDFQQ 439            GQQGVQ IQK+V V TLW+GA LVI   L++GQLIAFNNLSGQV APVIRLAQLWQDFQQ Sbjct: 377 GQQGVQFIQKVVNVITLWLGAMLVISGDLSIGQLIAFNNLSGQVIAPVIRLAQLWQDFQQ 436 Query: 440 VGISVAPLGDILNAPTENASSHLALPDIRGEITFEHVDFRYKADGRLILQDLNLRIRAGE 499            VGISV RLGD+LN+PTE+    LALP+I+G+ITF ++ FRYX D  +IL D+NL I+ GE Sbjct: 437 VGISVTRLGDVLNSPTESYQGKLALPEIKGDITFRNIRFRYXPDAPVILNDVNLSIQQGE 496 Query: 500 VLGIVGRSGSGKSTLTKLVQRLYVPAOGRVLVDGNDLALAAPAWLRRQVGVVLQENVLLN 559            V+GIVGRSGSGKSTLTKL+QR Y+P  G+VL+DG+DLALA P WLRRQVGVVLQ+NVLLN Sbjct: 497 VIGIVGRSGSGKSTLTKLIQRFYIPENGQVLIDGHDLALADPNWLRRQVGVVLQDNVLLN 556 Query: 560 RSIRDNIALTDTGMPLERIIEAAKLAGAIIEFINELPEGYGTVVGEQGNLSGGQRQRIAI 619            RSIRDNIAL D GMP+E+I+ AAKLAGAHEFI EL EGY T+VGEQGAGLSGGQRQRIAI Sbjct: 557 RSIRDNIALADPGMPMEKIVHAAKLAGAHEFISELREGYNTIVGEOGAGLSGGQRQRIAI 616 Query: 620 ARALITNPRILIFDEATSALDYESERAIMQNMQAICANRTVLIIAHRLSTVKTAHRIIAM 679            ARAL+ NP+ILIFDEATSALDYESE IM+NM  IC  RTV+IIAHRLSTVK A RII M Sbjct: 617 ARALVNNPKILIFDEATSALDYESEHIIMRNMHQICKGRTVIIIAHRLSTVKNADRIIVM 676 Query: 680 DKGRIVEAGTQQELLAKPNGYYRYLYDLQN 709            +KG+IVE G  +ELLA PNG Y YL+ LQ+ Sbjct: 677 EKGQIVEQGKHKELLADPNGLYHYLHQLQS 706 Homology with the HlyB Leucotoxin Secretion ATP-Binding Protein of Haemophilus actinomycetemcomitans (Accession Number X53955)

ORF39 and HlyB protein show 71% and 69% amino acid identity in 167 and 55 overlap at the N- and C-terminal regions, respectively: Orf39   1 KFDFTWFIPAVIKYRRXXXXXXXXXXXXXXXXXITPLFFQVVMDKVLVHRGFXXXXXXXX 60     KFDFTWFIPAVIKYR+                 ITPLFFQVVMDKVLVHRGF HlyB 137 KFDFTWFIPAVIKYRKIFIETLIVSIFLQIFALITPLFFQVVMDKVLVHRGFSTLNVITV 196 Orf39  61 XXXXXXXFEIVLGGLRTYLFAHTTSRIDVELGARLFRHLLSLPLSYFEHRRVGDTVARVR 120            FEI+LGGLRTY+FAH+TSRIDVELGARLFMLL+LP+SYFE RRVGDTVARVR HlyB 197 ALAIVVLFEIILGGLRTYVFAHSTSRIDVELGARLFRHLLALPISYFEARRVGDTVARVR 256 Orf39 121 ELEQIRNFLTGQALTSVLDLAFSFIFLAVMWYYSSTLTWVVLASLIC 167     EL+QIRNFLTGQALTS+LDL FSFIF AVMWYYS  LT VVL SL C HlyB 257 ELDQIRNFLTGQALTSILDLLFSFIFFAVMWYYSPKLTLVVLGSLPC 303                                 // Orf39 166 ICANRTVLIIAHRLSTVKTAHRIIAMDKGRIVEAGTQQELLANXNGYYRYLYDLQ 220     IC NRTVLIIAHRLSTVK A RII MDKG I+E G  QELL +  G Y YL+ LQ HlyB 651 ICQNRTVLIIAHRLSTVKNADRIIVMDKGEIIEQGKHQELLKDEKGLYSYLHQLQ 705

Based on this analysis, it is predicted that this protein from N. meningitidis, and its epitopes, could be useful antigens for vaccines or diagnostics.

Example 7

The following partial DNA sequence was identified in N. meningitidis <SEQ ID 31> 1 ATGAAATACT TGATCCGCAC CGCCTTACTC GCAGTCGCAG CCGCCGGCAT 51 CTACGCCTGC CAACCGCAAT CCGAAGCCGC AGTGCAAGTC AAGGCTGAAA 101 ACAGCCTGAC CGCTATGCGC TTAGCCGTCG CCGACAAACA GGCAGAGATT 151 GACGGGTTGA ACGCCCAAAk sGACGCCGAA ATCAGA...

This corresponds to the amino acid sequence SEQ ID 32; ORF52>: 1 MKYLIRTALL AVAAAGIYAC QPQSEAAVQV KAZNSLTANR LAVADKQAEI 51 DGLNAQXDAE IR..

Further work revealed the complete nucleotide sequence <SEQ ID 33>: 1 ATGAAATACT TGATCCGCAC CGCCTTACTC GCAGTCGCAG CCGCCGGCAT 51 CTACGCCTGC CAACCGCAAT CCGAAGCCGC AGTGCAAGTC AAGGCTGAAA 101 ACAGCCTGAC CGCTATGCGC TTAGCCGTCG CCGACAAACA GGCAGAGATT 151 GACGGGTTGA ACGCCCAAAT CGACGCCGAA ATCAGACAAC GCGAAGCCGA 201 AGAATTGAAA GACTACCGAT GGATACACGG CGACGCGGAA GTGCCGGAGC 251 TGGAAAAATG A

This corresponds to the amino acid sequence <SEQ ID 34; ORF52-1>: 1 MKYLIRTALL AVAAAGIYAC QPQSEAAVQV KAENSLTAMR LAVADKQAEI 51 DGLNAQIDAE IRQREAEELK DThWIHGDAE VPELEK

Computer analysis of this amino acid sequence predicts a prokaryotic membrane lipoprotein lipid attachment site (underlined).

ORF52-1 (7 kDa) was cloned in the pGex vectors and expressed in E. coli, as described above. The products of protein expression and purification were analyzed by SDS-PAGE. FIG. 4A shows the results of affinity purification of the GST-fusion. FIG. 4B shows plots of hydrophilicity, antigenic index, and AMPHI regions for ORF52-1.

Based on this analysis, it is predicted that this protein from N. meningitidis, and its epitopes, could be useful antigens for vaccines or diagnostics.

Example 8

The following DNA sequence was identified in N. meningitidis <SEQ ID 35> 1 ATGGTTATCG GAATATTACT CGCATCAAGC AAGCATGCTC TTGTCATTAC 51 TCTATTGTTA AATCCCGTCT TCCATGCATC CAGTTGCGTA TCGCGTTSGG 101 CAATACGGAA TAAAATCTGC TGTTCTGCTT TGGCTAAATT TGCCAAATTG 151 TTTATTGTTT CTTTAGGAGC AGCTTGCTTA GCCGCCTTCG CTTTCGACAA 201 CGCCCCCACA GGCGCTTCCC AAGCGTTGCC TTCCGTTACC GCACCCGTGG 251 CGATTCCCGC GCCCGCTTCG GCAGCCTGA

This corresponds to the amino acid sequence <SEQ ID 36; ORF56>: 1 MVIGILLASS KHALVITLLL NPVFHASSCV SRXAIRNKIC CSALAKFAKL 51 FIVSLGAACL AAFAFDNAPT GASQALPTVT APVAIPAPAS AA*

Further work revealed the complete nucleotide sequence <SEQ ID 37>: 1 ATGGCTTGTA CAGGTTTGAT GGTTTTTCCG TTAATGGTYA TCGGAATATT 51 ACTTGCATCA AGCAAGCCTG CTCCTTTCCT TACTCTATTG TTAAATCCCG 101 TCTTCCATGC ATCCAGTTGC GTATCGCGTT GGGCAATACG GAATAAAATC 151 TGCTGTTCTG CTTTGGCTAA ATTTGCCAAA TTGTTTATTG TTTCTTTAGG 201 AGCAGCTTGC TTAGCCGCCT TCGCTTTCGA CAACGCCCCC ACAGGCGCTT 251 CCCAAGCGTT GCCTACCGTT ACCGCACCCG TGGCGATTCC CGCGCCCGCT 301 TCGGCAGCCT GA

This corresponds to the amino acid sequence <SEQ ID 38; ORF56-1>: 1 MACTGLMVFP LNVZGILLAS SKPAPFLTLL LNPVFHASSC VSRWAIRNKI 51 CCSALAKFAK LFIVSLGAAC LAAFAFDNAP TGASQALPTV TAPVAIPAPA 101 SAA*

Computer analysis of this amino acid sequence predicts a leader peptide (underlined) and suggests that ORF56 might be a membrane or periplasmic protein.

Based on this analysis, it is predicted that this protein from N. meningitidis, and its epitopes, could be useful antigens for vaccines or diagnostics.

Example 9

The following partial DNA sequence was identified in N. meningitidis <SEQ ID 39> 1 ATGTTCAGTA TTTTAAATGT GTTTCTTCAT TGTATTCTGG CTTGTGTAGT 51 CTCTGGTGAG ACGCCTACTA TATTTGGTAT CCTTGCTCTT TTTTACTTAT 101 TGTATCTTTC TTATCTTGCT GTTTTTAAGA TTTTCTTTTC TTTTTTCTTA 151 GACAGAGTTT CACTCCGGTC TCCCAGGCTG GAGTGCAAAT GGCATGACCC 201 TTTGGCTCAC TGGCTCACGG CCACTTCTGC TATTCTGCCG CCTCAGCCTC 251 CAGGG...

This corresponds to the amino acid sequence <SEQ ID 40; ORF63>: 1 MFSILNVFLR CILACVVSGE TPTIFGILAL FYLLYLSYLA VFKIFFSFFL 51 DRVSLRSPRL ECKWNDPLAH WLTATSAILP PQPPG...

Computer analysis of this amino acid sequence predicts a transmembrane region.

Based on this analysis, it is predicted that this protein from N. meningitidis, and its epitopes, could be useful antigens for vaccines or diagnostics.

Example 10

The following partial DNA sequence was identified in N. meningitidis <SEQ ID 41> 1 ..GTGCGGACGT GGTTGGTTTT TTGGTTGCAG CGTTTGAAAT ACCCGTTGTT 51   GCTTTGGATT GCGGATATGT TGCTGTACCG GTTGTTGGGC GGCGCGGAAA 101   TCGAATGCGG CCGTTGCCCT GTGCCGCCGA TGACGGATTG GCAGCATTTT 151   TTGCCGGCGA TGGGAACGGT GTCGGCTTGG GTGGCGGTGA TTTGGGCATA 201   CCTGATGATT GAAAGTGAAA AAAACGGAAG ATATTGA

This corresponds to the amino acid sequence <SEQ ID 42; ORF69>: 1 ..VRTWLVFWLQ RLKYPLLLWI ADNLLYRLLG GAE1ECGRCP VPPMTDWQHF 51   LPANGTVSAW VAVIWAYLMI ESEKNGRY*

Computer analysis of this amino acid sequence predicts a transmembrane region.

A corresponding ORF from strain A of N. meningitidis was also identified:

Homology with a Predicted ORF from N. meningitidis (Strain A)

ORF69 shows 96.2% identity over a 78 aa overlap with an ORF (ORF69a) from strain A of N. meningitidis:         10        20        30        40        50        60 orf69.pep VRTWLVFWLQRLKYPLLLWIADMLLYRLLGGAEIECGRCPVPPMTDWQHFLPAMGTVSAW |||||||||||||||||| |||||||||||||||||||||||||||||||||:||||:|| orf69a VRTWLVFWLQRLKYPLLLCIADMLLYRLLGGAEIECGRCPVPPMTDWQHFLPTMGTVAAW         10        20        30        40        50        60         70        79 orf69.pep VAVIWAYLMIESEKNGRYX ||||||||||||||||||| orf69a VAVIWAYLMIESEKNGRYX         70        79

The ORF69a nucleotide sequence <SEQ ID 43> is: 1 GTGCGGACGT GGTTGGTTTT TTGGTTGCAG CGTTTGAAAT ACCCGTTGTT 51 GCTTTGTATT GCGGATATGC TGCTGTACCG GTTGTTGGGC GGCGCGGAAA 101 TCGAATGCGG CCGTTGCCCT GTACCGCCGA TGACGGATTG GCAGCATTTT 151 TTGCCGACGA TGGGAACGGT GGCGGCTTGG GTGGCGGTGA TTTGGGCATA 201 CCTGATGATT GAAAGTGAAA AAAACGGAAG ATATTGA

This encodes a protein having amino acid sequence <SEQ ID 44>: 1 VRTWLVFWLQ RLKYPLLLCI ADMLLYRLLG GAEIECGRCP VPPNTDWQHF 51 LPTMGTVAAW VAVIWAYLMI ESEKNGRY*

Based on this analysis, it is predicted that this protein from N. meningitidis, and its epitopes, could be useful antigens for vaccines or diagnostics.

Example 11

The following DNA sequence was identified in N. meningitidis <SEQ ID 45> 1 ATGTTTCAAA ATTTTGATTT GGGCGTGTTC CTGCTTGCCG TCCTCCCCGT 51 GCTGCCCTCC ATTACCGTCT CGCACGTGGC GCGCGGCTAT ACGGCGCGCT 101 ACTGGGGAGA CAACACTGCC GAACAATACG GCAGGCTGAC ACTGAACCCC 151 CTGCCCCATA TCGATTTGGT CGGCACAATC ATCgTACCGC TGCTTACTTT 201 GATGTTCACG CCCTTCCTGT TCGGCTGGGC GCGTCCGATT CCTATCGATT 251 CGCGCAACTT CCGCAACCCG cGCCTTGCCT GGCGTTGCGT TGCCGCGTCC 301 GGCCCGCTGT CGAATCTAGC GATGGCTGTw CTGTGGGGCG TGGTTTTGGT 351 GCTGACTCCG TATGTCGGCG GGGCGTATCA GATGCCGTTG GCTCAAATGG 401 CAAACTACGG TATTCTGATC AATGCGATTC TGTTCGCGCT CAACATCATC 451 CCCATCCTGC CTTGGGACGG CGGCATTTTC ATCGACACCT TCCTGTCGGC 501 GAAATATTCG CAAGCGTTCC GCAAAATCGA ACCTTATGGG ACGTGGATTA 551 TCCTACTGCT GATGCTGACC SGGGTTTTGG GTGCGTTTAT wGCACCGATT 601 sTGCGGmTGc GTGATTGCrT TTGTGCAGAT GTwCGTCTGA CTGGCTTTCA 651 GACGGCATAA

This corresponds to the amino acid sequence <SEQ ID 46; ORF77>: 1 MFQNFDLGVF LLAVLPVLPS ITVSNVARGY TARYWGDNTA EQYGRLTLNP 51 LPHIDLVGTI IVPLLTLMFT PFLFGWPRPI PIDSRNFRNP RLAWRCVAAS 101 GPLSNLAMAV LWGVVLVLTP YVGGAYQMPL AQMANYGILI NAILFPLNII 151 PILPWDGGIF IDTFLSAKYS QAFRKIEPYG TWIILLLMLT XVLGAFIAPI 201 XRXRDCXCAD VRLTGFQTA*

Further work revealed the complete nucleotide sequence <SEQ ID 47>: 1 ATGTTTCAAA ATTTTGATTT GGGCGTGTTT CTGCTTGCCG TCCTGCCCGT 51 GCTGCTCTCC ATTACCGTCA GGGAGGTGGC GCGCGGCTAT ACGGCGCGCT 101 ACTGGGGAGA CAACACTGCC GAACAATACG GCAGGCTGAC ACTGAACCCC 151 CTGCCCCATA TCGATTTGGT CGGCACAATC ATCGTACCGC TGCTTACTTT 201 GATGTTCACG CCCTTCCTGT TCGGCTGGGC GCGTCCGATT CCTATCGATT 251 CGCGCAACTT CCGCAACCCG CGCCTTGCCT GGCGTTGCGT TGCCGCGTCC 301 GGCCCGCTGT CGAATCTAGC GATGGCTGTT CTGTGGGGCG TGGTTTTGGT 351 GCTGACTCCG TATGTCGGCG GGGCGTATCA GATGCCGTTG GCTCAAATGG 401 CAAACTACGG TATTCTGATC AATGCGATTC TGTTCGCGCT CAACATCATC 451 CCCATCCTGC CTTGGGACGG CGGCATTTTC ATCGACACCT TCCTGTCGGC 501 GAAATATTCG CAAGCGTTCC GCAAAATCGA ACCTTATGGG ACGTGGATTA 551 TCCTACTGCT GATGCTGACC GGGGTTTTGG GTGCGTTTAT TGCACCGATT 601 GTGCGGCTGG TGATTGCGTT TGTGCAGATG TTCGTCTGA

This corresponds to the amino acid sequence <SEQ ID 48; ORF77-1>: 1 MFQNFDLGVF LLAVLPVLLS ITVREVARGY TARYWGDNTA EQYGRLTLNP 51 LPHIDLVGTI IVPLLTLMFT PFLFGWARPI PIDSRNFRNP RLAWRCVAAS 101 GPLSNLAMAV LWGVVLVLTP YVGGAYQMPL AQMANYGILI NAILFALNII 151 PILPWDGGIF IDTFLSAXYS QAYRRIEPYG TWIILLLNLT GVLGAFIAPI 201 VRLVIAFVQH FV*

Computer analysis of this amino acid sequence reveals a putative leader sequence and several transmembrane domains.

A corresponding ORF from strain A of N. meningitidis was also identified:

Homology with a Predicted ORF from N. meningitidis (Strain A)

ORF77 shows 96.5% identity over a 173 aa overlap with an ORF (ORF77a) from strain A of N. meningitidis:         10        20        30        40        50        60 ort77.pep MFQNFDLGVFLLAVLPVLPSITVSHVARGYTARYWGDNTAEQYGRLTLNPLPHIDLVGTI                            ||||||||||||||||||||||||||||||||| orf77a                            RGYTARYWGDNTAEQYGRLTLNPLPHIDLVGTI                                    10        20        30         70        80        90       100       110        120 orf77.pep IVPLLTLMFTPFLFGWARPIPIDSRNFRNPRLAWRCVAASGPLSNLAMAVLWGVVLVLTP |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| orf77a IVPLLTLMFTPFLFGWARPIPIDSRNFRNPRLAWRCVAASGPLSNLAMAVLWGVVLVLTP      40        50        60        70        80        90        130       140       150       160       170       180 orf77.pep YVGGAYQMPLAQMANYGILINAILFALNIIPILPWDGGIFIDTFLSAKYSQAFRKIEPYG |||||||||||||||| ||||||| ||||||||||||||||||||||| ||||||||||| orf77a YVGGAYQMPLAQMANYXILINAILXALNIIPILPWDGGIFIDTFLSAKXSQAFRKIEPYG     100       110       120       130       140       150        190       200       210       220 orf77.pep TWIILLLMLTGVLGAFIAPIVRLVIAFVQMFVX |||| |||||||||| |||||:||||||||||| orf77a TWIIXLLMLTGVLGAXIAPIVQLVIAFVQMFVX     160       170       180

ORF77-1 and ORF77a show 96.8% identity in 185 aa overlap:         10        20        30        40        50        60 orf77-1.pep MFQNFDLGVFLLAVLPVLLSITVREVARGYTARYWGDNTAEQYGRLTLNPLPHIDLVGTI                            ||||||||||||||||||||||||||||||||| orf77a                            RGYTARYWGDNTAEQYGRLTLNPLPHIDLVGTI                                    10        20        30         70        80        90       100       110       120 orf77-1.pep IVPLLTLMFTPFLFGWARPIPIDSRNFRNPRLAWRCVAASGPLSNLAMAVLWGVVLVLTP |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| orf77a IVPLLTLMFTPFLFGWARPIPIDSRNFRNPRLAWRCVAASGPLSNLAMAVLWGVVLVLTP      40        50        60        70        80        90        130       140       150       160       170       180 orf77-1.pep YVGGAYQMPLAQMANYGILINAILFALNIIPILPWDGGIFIDTFLSAKYSQAFRKIEPYG |||||||||||||||| ||||||| ||||||||||||||||||||||| ||||||||||| orf77a YVGGAYQMPLAQMANYXILINAILXALNIIPILPWDGGIFIDTFLSAKXSQAFRKIEPYG     100       110       120       130       140       150        190       200       210 orf77-1.pep TWIILLLMLTGVLGAFIAPIVRLVIAFVQMFVX |||| |||||||||| |||||:||||||||||| orf77a TWIIXLLMLTGVLGAXIAPIVQLVIAFVQMFVX     160       170       180

A partial ORF77a nucleotide sequence <SEQ ID 49> was identified: 1 ..CGCGGCTATA CAGCGCGCTA CTGGGGTGAC AACACTGCCG AACAATACGG 51   CAGGCTGACA CTGAACCCCC TGCCCCATAT CGATTTGGTC GGCACAATCA 101   TCGTACCGCT GCTTACTTTG ATGTTTACGC CCTTCCTGTT CGGCTGGGCG 151   CGTCCGATTC CTATCGATTC GCGCAACTTC CGCAACCCGC GCCTTGCCTG 201   GCGTTGCGTT GCCGCGTCCG GCCCGCTGTC GAATCTGGCG ATGGCTGTTC 251   TGTGGGGCGT GGTTTTGGTG CTGACTCCGT ATGTCGGTGG GGCGTATCAG 301   ATGCCGTTGG CNCAAATGGC AAACTACNNN ATTCTGATCA ATGCGATTCT 351   GTNCGCGCTC AACATCATCC CCATCCTGCC TTGGGACGGC GGCATTTTCA 401   TCGACACCTT CCTGTCGGCN AAATANTCGC AAGCGTTCCG CAAAATCGAA 451   CCTTATGGGA CGTGGATTAT CCNGCTGCTT ATGCTGACCG GGGTTTTGGG 501   TGCGTNTATT GCACCGATTG TGCAGCTGGT GATTGCGTTT GTGCAGATGT 551   TCGTCTGA

This encodes a protein having amino acid sequence <SEQ ID 50>: 1 ..RGYTARYWGD NTAEQYGRLT LNPLPHIDLV GTIIVPLLTL MFTPFLFGWA 51   RPIPIDSRNF RNPRLAWRCV AASGFLSNLA MAVLWGVVLV LTPYVGGAYQ 101   MPLAQNANYX ILINAILXAL NIIPILPWDG GIFIDTFLSA KXSQAFRKIE 151   PYGTWIIXLL MLTGVLGAXI APIVQLVIAF VQNFV*

Based on this analysis, it is predicted that this protein from N. meningitidis, and its epitopes, could be useful antigens for vaccines or diagnostics.

Example 12

The following partial DNA sequence was identified in N. meningitidis SEQ ID 51> 1 ATGAACCTGA TTTCACGTTA CATCATCCGT CAAATGGCGG TTATGGCGGT 51 TTACGCGCTC CTTGCCTTCC TCGCTTTGTA CAGCTTTTTT GAAATCCTGT 101 ACGAAACCGG CAACCTCGGC AAAGGCAGTT ACGGCATATG GGAAATGCTG 151 GGCTACACCG CCCTCAAAAT GCCCGCCCGC GCCTACGAAC TGATTCCCCT 201 CGCCGTCCTT ATCGGCGGAC TGGTCTCCCT CAGCCAGCTT GCCGCCGGCA 251 GCGAACTGAC CGTCATCAAA GCCAGCGGCA TGAGCACCAA AAAGCTGCTG 301 TTGATTCTGT CGCAGTTCGG TTTTATTTTT GCTATTGCCA CCGTCGCGCT 351 CGGCGAATGG GTTGCGCCCA CACTGAGCCA AAAAGCCGAA AACATCAAAG 401 CCGCCGCCAT CAACGGCAAA ATCAACACCG GCAATACCGG CCTTTGGCTG 451 AAAGAAAAAA ACAGCGTGAT CAATGTGCGC GAAATGTTGC CCGACCAT..

This corresponds to the amino acid sequence SEQ ID 52; ORF112>: 1 HNLISRYIIR QMAVMAVYAL LAFLALYSFF EILYETGNLG KGSYGIWEML 51 GYTALIQPAR AYELIPLAVL IGGLVSLSQL AAGSELTVIK ASGNSTKKLL 101 LILSQFGFIF AIATVPLGEW VAPTLSQKAE NIKAAAINGK ISTGNTGLWL 151 KEKNSVINVR EHLPDH...

Further work revealed further partial nucleotide sequence <SEQ ID 53>: 1 ATGAACCTGA TTTCACGTTA CATCATCCGT CAAATGGCGG TTATGGCGGT 51 TTACGCGCTC CTTGCCTTCC TCGCTTTGTA CAGCTTTTTT GAAATCCTGT 101 ACGAAACCGG CAACCTCGGC AAAGGCAGTT ACGGCATATG GGAAATGCTG 151 GGCTACACCG CCCTCAAAAT GCCCGCCCGC GCCTACGAAC TGATTCCCCT 201 CGCCGTCCTT ATCGGCGGAC TGGTCTCCCT CAGCCAGCTT GCCGCCGGCA 251 GCGAACTGAC CGTCATCAAA GCCAGCGGCA TGAGCACCAA AAAGCTGCTG 301 TTGATTCTGT CGCAGTTCGG TTTTATTTTT GCTATTGCCA CCGTCGCGCT 351 CGGCGAATGG GTTGCGCCCA CACTGAGCCA AAAAGCCGAA AACATCAAAG 401 CCGCCGCCAT CAACGGCAAA ATCAGCACCG GCAATACCGG CCTTGCTCTG 451 AAAGAAAAAA ACAGCTTAAT CAATGTGCGC GAAATGTTGC CCGACCATAC 501 GCTTTTGGGC ATCAAAATTT GGGCGCGCAA CGATAAAAAC GAATTGGCAG 551 AGGCAGTGGA AGCCGATTCC GCCGTTTTGA ACAGCGACGG CAGTTGGCAG 601 TTGAAAAACA TCCGCCGCAG CACGCTTGGC GAAGACAAAG TCGAGGTCTC 651 TATTGCGGCT GAAGAAAACT GGCCGATTTC CGTCAAACGC AACCTGATGG 701 ACGTATTGCT CGTCAAACCC GACCAAATGT CCGTCGGCGA ACTGACCACC 751 TACATCCGCC ACCTCCAAAA CAACAGCCAA AACACCCGAA TCTACGCCAT 601 CGCATGGTGG CGCAAATTGG TTTACCCCGC CGCAGCCTGG GTGATGGCGC 851 TCGTCGCCTT TGCCTTTACC CCGCAAACCA CCCGCCACGG CAATATGGGC 901 TTAAAACTCT TCGGCGGCAT CTGTSTCGGA TTGCTGTTCC ACCTTGCCGG 951 ACGGCTCTTT GGGTTTACCA GCCAACTCGG...

This corresponds to the amino acid sequence <SEQ ID 54; ORF112-1>: 1 MNLISRYIIR QMAVMAVYAL LAFLALYSFF EILYETGNLG KGSYGIWEHL 51 GYTALKMPAR AYELIPLAVL IGGLVSLSQL AAGSELTVIK ASGMSTKKLL 101 LILSQFGFIF AIATVALGEW VAPTLSQKAE NIKAAAINGK ISTGNTGLWL 151 KEKNSXINVR EHLPDHTLLG IKIWARNDKN ELAEAVEADS AVLNSDGSWQ 201 LKNIRRSTLG EDKVEVSIAA EENWPISVKR NLTDVLLVKP DQMSVGELTT 251 YIRHLONNSQ NTRIYAIAWW RKLVYPAAAW VMALVAFAFT PQTTRHGNMG 301 LKLFGGICXG LLFHLAGRLF GFTSQL...

Computer analysis of this amino acid sequence predicts two transmembrane domains.

A corresponding ORF from strain A of N. meningitidis was also identified:

Homology with a Predicted ORF from N. meningitidis (Strain A)

ORF112 shows 96.4% identity over a 166 aa overlap with an ORF (ORF112a) from strain A of N. meningitidis:         10        20        30        40        50        60 orf112.pep MNLISRYIIRQMAVMAVYALLAFLALYSFFEILYETGNLGKGSYGIWEMLGYTALKMPAR |||||||||||||||||||||||||||||||||||||||||||||||| ||||||| || orf112a MNLISRYIIRQMAVMAVYALLAFLALYSFFEILYETGNLGKGSYGIWEMXGYTALKMXAR         10        20        30        40        50        60         70        80        90       100       110       120 orf112.pep AYELIPLAVLIGGLVSLSQLAAGSELTVIKASGMSTKKLLLILSQFGFIFAIATVALGEW ||||:||||||||||| |||||||||:||||||||||||||||||||||||||||||||| orf112a AYELMPLAVLIGGLVSXSQLAAGSELTVIKASGMSXKKLLLILSQFGFIFAIATVALGEW         70        80        90       100       110       120        130       140       150       160 orf112.pep VAPTLSQKAENIKAAAINGKISTGNTGLWLKEKNSVINVREMLPDH |||||||||||||||||||||||||||||||||||:|||||||||| orf112a VAPTLSQKAENIKAAAINGKISTGNTGLWLKEKNSIINVREMLPDHTLLGIKIWARNDKN        130       140       150       160 orf112a ELAEAVEADSAVLNSDGSWQLKNIRRSTLGEDKVEVSIAAEEXWPISVKRNLMDVLLVKP        190       200       210       220       230       240

A partial ORF112a nucleotide sequence <SEQ ID 55> was identified: 1 ATGAACCTGA TTTCACGTTA CATCATCCGT CAAATGGCGG TTATGGCGGT 51 TTACGCGCTC CTTGCCTTCC TCGCTTTGTA CAGCTTTTTT GAAATCCTGT 101 ACGAAACCGG CAACCTCGGC AAAGGCAGTT ACGGCATATG GGAAATGNTG 151 GGNTACACCG CCCTCAAAAT GNCCGCCCGC GCCTACGAAC TGATGCCCCT 201 CGCCGTCCTT ATCGGCGGAC TGGTCTCTNT CAGCCAGCTT GCCGCCGGCA 251 GCGAACTGAN CGTCATCAAA GCCAGCGGCA TGAGCACCAA AAAGCTGCTG 301 TTGATTCTGT CGCAGTTCGG TTTTATTTTT GCTATTGCCA CCGTCGCGCT 351 CGGCGAATGG GTTGCGCCCA CACTGAGCCA AAAAGCCGAA AACATCAAAG 401 CCGCGGCCAT CAACGGCAAA ATCAGTACCG GCAATACCGG CCTTTGGCTG 451 AAAGAAAAAA ACAGCATTAT CAATGTGCGC GAAATGTTGC CCGACCATAC 501 CCTGCTGGGC ATTAAAATCT GGGCCCGCAA CGATAAAAAC GAACTGGCAG 551 AGGCAGTGGA AGCCGATTCC GCCGTTTTGA ACAGCGACGG CAGTTGGCAG 601 TTGAAAAACA TCCGCCGCAG CACGCTTGGC GAAGACAAAG TCGAGGTCTC 651 TATTGCGGCT GAAGAAAANT GGCCGATTTC CGTCAAACGC AACCTGATGG 701 ACGTATTGCT CGTCAAACCC GACCAAATGT CCGTCGGCGA ACTGACCACC 751 TACATCCGCC ACCTCCAAAN NNACAGCCAA AACACCCGAA TCTACGCCAT 801 CGCATGGTGG CGCAAATTGG TTTACCCCGC CGCAGCCTGG GTGATGGCGC 851 TCGTCGCCTT TGCCTTTACC CCGCAAACCA CCCGCCACGG CAATATGGGC 901 TTAAAANTCT TCGGCGGCAT CTGTCTCGGP TTGCTGTTCC ACCTTGCCGG 951 NCGGCTCTTC NGGTTTACCA GCCAACTCTA CGGCATCCCG CCCTTCCTCG 1001 NCGGCGCACT ACCTACCATA GCCTTCGCCT TGCTCGCCGT TTGGCTGATA 1051 CGCAAACAGG AAAAACGCTA A

This encodes a protein having amino acid sequence <SEQ ID 56>: 1 MNLISRYIIR QMAVMAVYAL LAFLALYSFF EILYETGNLG KGSYGIWEMK 51 GYTALKMXAR AYELMPLAVL IGGLVSXSQL AAGSELXVIX ASGNSTKKLL 101 LILSQFGFIF AIATVALGEV VAPTLSQKAE NIKAAAINGK ISTGNTGLWL 151 KEKNSIINVR EMLPDHTLLG IKIWAIWDKN ELAEAVFADS AVLNSDGSWQ 201 LKNIRRSTLG EDKVEVSIAA EEXWPISVKR NLMDVLLVKP DQMSVGELTT 251 YIRHLQXXSQ NTRIYAIAWW RKLVYPAAAW VMALVAFAFT PQTTRHGNMG 301 LKXFGGICLG LLFHLAGRLF XFTSQLYGIP PFLXGALPTI AFALLAVWLI 351 RKQEKR*

ORF112a and ORF112-1 show 96.3% identity in 326 aa overlap: orf112a.pep MNLISRYIIRQMAVMAVYALLAFLALYSFFEILYETGNLGKGSYGIWEMXGYTALKMXAR ||||||||||||||||||||||||||||||||||||||||||||||||| ||||||| || orf112-1 MNLISRYIIRQMAVMAVYALLAFLALYSFFEILYETGNLGKGSYGIWEMLGYTALKMXAR orf112a.pep AYEIIPLAVLIGGLVSXSQLAAGSELXVIKASGHSTKKLLLILSQFGFIFAIATVALGEW ||||:||||||||||| |||||||||:||||||||||||||||||||||||||||||||| orf112-1 AYEIIPLAVLIGGLVSLSQLAAGSELTVIKASGHSTKKLLLILSQFGFIFAIATVALGEW orf112a.pep VAPTLSQKAENIKAAAINGKISTGNTGLWLKEKNSIINVREMLPDHTLLGIKIWARNDKN ||||||||||||||||||||||||||||||||||| |||||||||||||||||||||||| orf112-1 VAPTLSQKAENIKAAAINGKISTGNTGLWLKEKNSXINVREMLPDHTLLGIKIWARNDKN orf112a.pep ELAEAVEADSAVLNSDGSWQLKNIRRSTLGEDKVEVSIAAEEXWPISVKRNLMDVLLVKP |||||||||||||||||||||||||||||||||||||||||| ||||||||||||||||| orf112-1 ELAEAVEADSAVLNSDGSWQLKNIRRSTLGEDKVEVSIAAEENWPISVKRNLMDVLLVKP orf112a.pep DQMSVGELTTYIRHLQXXSQNTRIYAIAWWRKLVYPAAAWVMALVAFAFTPQTTRHGNMG ||||||||||||||||  |||||||||||||||||||||||||||||||||||||||||| orf112-1 DQMSVGELTTYIRHLQXXSQNTRIYAIAWWRKLVYPAAAWVMALVAFAFTPQTTRHGNMG orf112a.pep LKXFGGICLGLLFHLAGRLFXFTSQLYGIPPFLXGALPTIAFALLAVWLIRKQEXRX || ||||| ||||||||||| ||||| orf112-1 LKLFGGICXGLLFHLAGRLFGFTSQL

Based on this analysis, it is predicted that this protein from N. meningitidis, and its epitopes, could be useful antigens for vaccines or diagnostics.

Example 13

The following partial DNA sequence was identified in N. meningitidis <SEQ ID 57> 1 ..GCAGTAGCCG AAACTGCCAA CAGCCAGGGC AAAGGTAAAC AGGCAGGCAG 51   TTCGGTTTCT GTTTCACTGA AAACTTCAGG CGACCTTTGC GGCAAACTCA 101   AAACCACCCT TAAAACTTTG GTCTGCTCTT TGGTTTCCCT GAGTATGGTA 151   TTGCCTGCCC ATGCCCAAAT TACCACCGAC AAATCACCAC CTAAAAACCA 201   GCAGGTCGTT ATCCTTAAAA CCAACACTGG TGCCCCCTTG GTGAATATCC 251   AAACTCCGAA TGGACGCGGA TTGAGCCACA ACCGCTA.TA CGCATTTGAT 301   GTTGACAACA AAGGGGCAGT GTTAAACAAC GACCGTAACA ATAATCCGTT 351   TGTGGTCAAA GGCAGTGCGC AATTGATTTT GAACGAGGTA CGCGGTACGG 401   CTAGCAAACT CAACGGCATC GTTACCGTAG GCGGTCAAAA GGCCGACGTG 451   ATTATTGCCA ACCCCAACGG CATTACCGTT AATGGCGGCG GCTTTAAAAA 501   TGTCGGTCGG GGCATCTTAA CTACCGGTGC GCCCCAAATC GGCAAAGACG 551   GTGCACTGAC AGGATTTGAT GTGCGTCAAG GCACATTGGA CCGTAGTAGC 601   AGCAGGTTGG AATGATAAAG GCGGAGCmrm yTACACCGGG GTACTTGCTC 651   GTGCAGTTGC TTTGCAGGGG AAATTwmmGG GTAAA.AACT GGCGGTTTCT 701   ACCGGTCCTC AGAAAGTAGA TTACGCCAGC GGCGAAATCA GTGCAGGTAC 751   GGCAGCGGGT ACGAAACCGA cTATTGCCCT TGATACTGCC GCACTGGGCG 901   GTATGTACGC CGACAGCATC ACACTGATTG CCAATGAAAA AGGCGTAGGC 951   GTCTAA

This corresponds to the amino acid sequence <SEQ ID 58; ORF114>: 1 ..AVAETANSQG KGKQAGSSVS VSLKTSGDLC GKLKTTLKTL VCSLVSLSHV 51   LPAHAQITTD KSAPKNQQVV ILKTNTGAPL VNIQTPNGRG LSHNRXYAFD 101   VDNKGAVLNN DRNNNPFVVK GSAQLILNEV RGTASKLNGI VTVGGQKADV 151   IIANPNGITV NGGGFXNVGR GILTTGAPQI GKDGALTGFD VVKAHVTVXA 201   AGWNDKGGAX YTGVLAPAVA LQGKXXGLXL AVSTGPQKVD YASGEISAGT 251   AAGTKPTIAL DTAALGGNYA DSITLIANEX GVGV*

Further work revealed the complete nucleotide sequence SEQ ID 59>: 1 ATGAATAAAG GTTTACATCG CATTATCTTT AGTAAAAAGC ACAGCACCAT 51 GGTTGCAGTA GCCGAAACTG CCAACAGCCA GGGCAAAGGT AAACAGGCAG 101 GCAGTTCGGT TTCTGTTTCA CTGAAAACTT CAGGCGACCT TTGCGGCAAA 151 CTCAAAACCA CCCTTAAAAC TTTGGTCTGC TTTTTGGTTT CCCTGAGTAT 201 GGTATTGCCT GCCCATGCCC AAATTACCAC CGACAAATCA GCACCTAAAA 251 ACCAGCAGGT CGTTATCCTT AAAACCAACA CTGGTGCCCC CTTGGTGAAT 301 ATCCAAACTC CGAATGGACG CGGATTGAGC CACAACCGCT ATACGCAGTT 351 TGATGTTGAC AACAAAGGGG CAGTGTTAAA CAACGACCGT AACAATAATC 401 CGTTTGTGGT CAAAGGCAGT GCGCAATTGA TTTTGAACGA GGTACGCGGT 451 ACGGCTAGCA AACTCAACGG CATCGTTACC GTAGGCGGTC AAAAGGCCGA 501 CGTGATTATT GCCAACCCCA ACGGCATTAC CGTTAATGGC GGCGGCTTTA 551 AAAATGTCGG TCGGGGCATC TTAACTACCG GTGCGCCCCA AATCGGCAAA 601 GACGGTGCAC TGACAGGATT TGATGTGCGT CAAGGCACAT TGACCGTAGG 651 AGCAGCAGGT TGGAATGATA AAGGCGGAGC CGACTACACC GGGGTACTTG 701 CTCGTGCAGT TGCTTTGCAG GGGAAATTAC AGGGTAAAAA CCTGGCGGTT 751 TCThCCGGTC CTCAGAAAGT AGATTACGCC AGCGGCGAAA TCAGTGCAGG 801 TACGGCAGCG GGTACGAAAC CGACTATTGC CCTTGATACT GCCGCACTGG 951 GCGGTATGTA CGCCGACACC ATCACACTGA TTGCCAATGA AAAAGGCGTA 901 GGCGTCAAAA ATGCCGGCAC ACTCGAAGCG GCCAAGCAAT TGATTGTGAC 951 TTCGTCAGGC CGCATTGAAA ACAGCGGCCG CATCGCCACC ACTGCCGACG 1001 GCACCGAAGC TTCACCGACT TATCTCTCCA TCGAAACCAC CGAAAAAGGA 1051 GCGGCAGGCA CATTTATCTC CAATGGTGGT CGGATCGAGA GCAAAGGCTT 1101 ATTGGTTATT GAGACGGGAG AAGATATCAG CTTGCGTAAC GGAGCCGTGG 1151 TGCAGAATAA CGGCAGTCTC CCAGCTACCA CGGTATTAAA TGCTGGTCAT 1201 AATTTGGTGA TTGAGAGCAA AACTAATGTG AACAATGCCA AAGGCCCGGC 1251 TACTCTGTCG GCCGACGGCC GTACCGTCAT CAAGGAGGCC AGTATTCAGA 1301 CTGGCACTAC CGTATACAGT TCCAGCAAAG GCAACGCCGA ATTAGGCAAT 1351 AACACACGCA TTACCGGGGC AGATGTTACC GTATTATCCA ACGGCACCAT 1401 CAGCAGTTCC GCCGTAATAG ATGCCAAAGA CACCGCACAC ATCGAAGCAG 1451 GCAAACCGCT TTCTTTGGAA GCTTCAACAG TTACCTCCGA TATCCGCTTA 1501 AACGGAGGCA GTATCAAGGG CGGCAAGCAG CTTGCTTTAC TGGCAGACGA 1551 TAACATTACT GCCAAAACTA CCAATCTGAA TACTCCCGGC AATCTGTATG 1601 TTCATACAGG TAAAGATCTG AATTIGAATG TTGATAAAGA TTTGTCIGCC 1651 GCCAGCATCC ATTTGAAATC GGATAACGCT GCCCATATTA CCGGCACCAG 1701 TAAAACCCTC ACTGCCTCAA AAGACATGGG TGTGGAGGCA GGCTCGCTGA 1751 ATGTTACCAA TACCAATCTG CGTACCAACT CGGGTAATCT GCACATTCAG 1801 GCAGCCAAAG GCAATATTCA GCTTCGCAAT ACCAAGCTGA ACGCAGCCAA 1851 GGCTCTCGAA ACCACCGCAT TGCAGGGCAA TATCGTTTCA GACGGCCTTC 1901 ATGCTGTTTC TGCAGACGGT CATGTATCCT TATCGGCCAA CGGTAATGCC 1951 GACTTTACCG GTCACAATAC CCTGACAGCC AAGGCCGATG TCAATGCAGG 2001 ATCGGTTGGT AAAGGCCGTC TGAAAGCAGA CAATACCAAT ATCACTTCAT 2051 CTTCAGGAGA TATTACGTTG GTTGCCGGCA ACGGTATTCA GCTTGGTGAC 2101 GGAAAACAAC GCAATTCAAT CAACGGAAAA CACATCAGCA TCAAAAACAA 2151 CGGTGGTAAT GCCGACTTAA AAAACCTTAA CGTCCATGCC AAAAGCGGGG 2201 CATTGAACAT TCATTCCGAC CGGGCATTGA GCATAGAAAA TACCAAGCTG 2251 GAGTCTACCC ATAATACGCA TCTTAATGCA CAACACGAAG GGGTAACGCT 2301 CAACCAAGTA GATGCCTACG CACACCGTCA TCTAAGCATT ACCGGCAGCC 2351 AGATTTGGCA AAACGACAAA CTGCCTTCTG CCAACAAGCT GGTGGCTAAC 2401 GGTGTATTGG CACTCAATGC GCGCTATTCC CAAATTGCCG ACAACACCAC 2451 GCTGAGAGCG GGTGCAATCA ACCTTATTGC CGGTACCGCC CTAGTCAAGC 2501 GCGGCAACAT CAATTGGAGT ACCGTTGCGA CCAAAACTTT GGAAGATAAT 2551 GCCGAATTAA AACCATTGGC CGGACGGCTG AATATTGAAG CAGGTAGCGG 2601 CACATTAACC ATCGAACCTG CCAACCGCAT CAGTGCGCAT ACCGACCTGA 2651 GCATCAAAAC AGGCGGAAAA TTGCTGTTGT CTGCAAAAGG AGGAAATGCA 2701 GGTGCGCCTA GTGCTCAAGT TTCCTCATTG GAAGCAAAAG GCAATATCCG 2751 TCTGGTTACA GGAGAAACAG ATTTAAGAGG TTCTAAAATT ACAGCCGGTA 2801 AAAACTTGGT TGTCGCCACC ACCAAAGGCA AGTTGAATAT CGAAGCCGTA 2951 AACAACTCAT TCAGCAATTA TTTTCCTACA CAAAAAGCGG CTGAACTCAA 2901 CCAAAAATCC AAAGAATTGG AACAGCAGAT TGCGCAGTTG AAAAAAAGCT 2951 CGCCTAAAAG CAAGCTGATT CCAACCCTGC AAGAAGAACG CGACCGTCTC 3001 GCTTTCTATA TTCAAGCCAT CAACAAGGAA GTTAAAGGTA AAAAACCCAA 3051 AGGCAAAGAA TACCTGCAAG CCAAGCTTTC TGCACAAAAT ATTGACTTGA 3101 TTTCCGCACA AGGCATCGAA ATCAGCGGTT CCGATATTAC CGCTTCCAAA 3151 AAACTGAACC TTCACGCCGC AGGCGTATTG CCAAAGGCAG CAGATTCAGA 3201 GGCGGCTGCT ATTCTGATTG ACGGCATAAC CGACCAATAT GAAATTGGCA 3251 AGCCCACCTA CAAGAGTCAC TACGACAAAG CTGCTCTGAA CAAGCCTTCA 3301 CGTTTGACCG GACGTACAGG GGTAAGTATT CATGCAGCTG CGGCACTCGA 3351 TGATGCACGT ATTATTATCG GTGCATCCGA AATCAAAGCT CCCTCAGGCA 3401 GCATAGACAT CAAAGCCCAT AGTGATATTG TACTGGAGGC TGGACAAAAC 3451 GATGCCTATA CCTTCTTAAA AACCAAAGGT AAAAGCGGCA AAATCATCAG 3501 AAAAACCAAG TTTACCAGCA CCCGCGACCA CCTGATTATG CCAGCCCCCG 3551 TCGAGCTGAC CGCCAACGGC ATAACGCTTC AGGCAGGCGG CAACATCGAA 3601 GCTAATACCA CCCGCTTCAA TGCCCCTGCA GGTAAAGTTA CCCTGGTTGC 3651 GGGTGAAGAG CTGCAACTGC TGGCAGAASA AGGCATCCAC AAGCACGAGT 3701 TGGATGTCCA AAAAAGCCGC CGCTTTATCG GCATCAAGGT AGGCAAGAGC 3751 AATTACAGTA AAAACGAACT GAACGAAACC AAATTGCCTG TCCGCGTCGT 3801 CGCCCAAACT GCAGCCACCC GTTCAGGCTG GGATACAGTG CTCGAAGGTA 3051 CCGAATTCAA AACCACGCTG GCCGGTGCGG ACATTCAGGC AGGTGTAGGC 3901 GAAAAAGCCC GTGCCGATGC GAAAATTATC CTCAAAGGCA TTGTGAACCG 3951 TATCCAGTCG GAAGAAAAAT TAGAAACCAA CTCAACCGTA TGGCAGAAAC 4001 AGGCCGGACG CGGCAGCACT ATCGAAACGC TGAAACTGCC CAGCTTCGAA 4051 AGCCCTACTC CGCCCAAACT GACCGCCCCC GGTGGCTATA TCGTCGACAT 4101 TCCGAAAGGC AATTTGAAAA CCGAAATCGA AAAGCTGGCC AAACAGCCCG 4151 AGTATGCCTA TCTGAAACAG CTCCAAGTAG CGAAAAACGT CAACTGGAAC 4201 CAGGTGCAAC TGGCTTACGA TAAATGGGAC TATAAGCAGG AAGGCTTAAC 4251 CAGAGCCGGT GCAGCGATTG TTACCATAAT CGTAACCGCA CTGACTTATG 4301 GATACGGCGC AACCGCAGCG GGCGGTGTAG CCGCTTCAGG AAGTAGTACA 4351 GCCGCAGCTG CCGGAACAGC CGCCACAACG ACAGCAGCAG CTACTACCGT 4401 TTCTACAGCG ACTGCCATGC AAACCGCTGC TTTAGCCTCC TTGTATAGCC 4451 AAGCAGCTGT ATCCATCATC AATAATAAAG GTGATGTCGG CAAAGCGTTG 4501 AAAGATCTCG GCACCAGTGA TACGGTCAAG CAGATTGTCA CTTCTGCCCT 4551 GACGGCGGGT GCATTAAATC AGATGGGCGC AGATATIGCC CAATTGAACA 4601 GCAAGGTAAG AACCGAACTG TTCAGCAGTA CGGGCAATCA AACTATTGCC 4651 AACCTTGGAG GCAGATTGGC TACCAATCTC AGTAATGCAG GTATCTCAGC 4701 TGGTATCAAT ACCGCCGTCA ACGGCGGCAG CCTGAAAGAC AACTTAGGCA 4751 ATGCCGCATT AGGAGCATTG GTTAATAGCT TCCAAGGAGA AGCCGCCAGC 4801 AAAATCAAAA CAACCTTCAG CGACGATTAT GTTGCCAAAC AGTTCGCCCA 4851 CGCTTTGGCT GGGTGTGTTA GCGGATTGGT ACAAGGAAAA TGTAAAGACG 4901 GGGCAATTGG CGCAGCAGTT GGGGAAATCG TAGCCGACTC CATGCTTGGC 4951 GGCAGAAACC CTGCTACACT CAGCGATGCG GAAAAGCATA AGGTTATCAG 5001 TTACTCGAAG ATTATTGCCG GCAGCGTGGC GGCACTCAAC GGCGGCGATG 5051 TGAATACTGC GGCGAATGCG GCTGAGGTGG CGGTAGTGAA TAATGCTTTG 5101 AATTTTGACA GTACCCCTAC CAATGCGAAA AAGCATCAAC CGCAGAAGCC 5151 CGACAAAACC GCACTGGAAA AAATTATCCA AGGTATTATG CCTGCACATG 5201 CAGCAGGTGC GATGACTAAT CCGCAGGATA AGGATGCTGC CATTTGGATA 5251 AGCAATATCC GTAATGGCAT CACAGGCCCG ATTGTGATTA CCAGCTATGG 5301 GGTTTATGCT GCAGGTTGGA CAGCTCCGCT GATCGGTACA GCGGGTAAAT 5351 TAGCTATCAG CACCTGCATG GCTAATCCTT CTGGTTGTAC TGTCATGGTC 5401 ACTCAGGCTG CCGAAGCGGG CGCGGGAATC GCCACGGGTG CGGTAACGGT 5451 AGGCAACGCT TGGGAAGCGC CTGTGGGGGC GTTGTCGAAA GCGAAGGCGG 5501 CCAAGCAGGC TATACCAACC CAGACAGTTA AAGAACTTGA TGGCTTACTA 5551 CAAGAATCAA AAAATATAGG TGGTGTAAAT ACACGAATAA ATATAGCGAA 5601 TAGTACTACT CGATATACAC CAATGAGACA AACGGGACAA CCGCTATCTG 5651 CTGGCTTTGA GCATGTFCTT GAGGGGGACT TCCATAGGCC TATTGCGAAT 5701 AACCGTTCAG TTTTTACCAT CTCCCCAAAT GAATTGAAGG TTATACTTCA 5751 AAGTAATAAA GTAGTTTCTT CTCCCGTATC GATGACTCCT GATGGCCAAT 5801 ATATGCGGAC TGTCGATGTA GGAAAAGTTA TTGGTACTAC TTCTATTAAA 5651 GAAGGTGGAC AACCCACAAC TACAATTAAA GTATTTACAG ATAAGTCAGG 5901 AAATTTGATT ACTACATACC CAGTAAAAGG AAACTAA

This corresponds to the amino acid sequence <SEQ ID 60; ORF114-1>: 1 HNKGLHRIIF SXKHSTMVAV AETANSQGKG KQAGSSVSVS LKTSGDLCGK 51 LKTTLKTLVC SLVSLSHVLP AHAQITTDKS APKNQQVVIL KFNTGAPLVN 101 IQTPNGRGLS HNRYTQFDVD NKGAVLNNDR NNNPFVVKGS AQLILNEVRG 151 TASKLNGIVT VGGQKADVII ANPNGITVNG GGFKNVGRGI LTTGAPQIGK 201 DGALTGFDVR QGTLTVGAAG WNDKGGADYT GVLAAAVALQ GKLQGKLLAV 251 STGFQKVDYA SGEISAGTAA GTKPTIALDT AALGGHYADS ITLIANEKGV 301 GVKNAGTLKA AXQLIVTSSG RIENSGRIAT TADGTFASPT YLSIETTEKG 351 AAGTFISNGG RIESKGLLVI ETGEDISLRN GAVVQINGSR PATTVLNAGH 401 HLVIESKTNV NNAKGFATLS ADGRTVIKEA SIQTGTTVYS SSKGTAELGN 451 NTRZTGADVT VLSNGTISSS AVIDAKOTAN IEAGKPLSLT ASTVTSDIRL 501 NGGSIKGGKQ LALLADDNIT AXTTNLNTPG NLYVHTGKDL NIMVDKDLSA 551 ASIHLKSDNA AHITGTSKTL TASKDMGVEA GSLNVTNTNL RTNSGNLHIQ 601 AAKGNIQLRN TKLNAAKALE TTALQGNIVS OGLHAVSADG HVSLLANGNA 651 DFTGHNTLTA KADVNAGSVG KGRLKADNTN ITSSSGDITL VAGNGIQLGD 701 GKQRNSINGK HISIKNNGGN ADLIQLNVHA KSGALNIHSD RALSIENTKL 751 ESTRNTHLNA QHERVTLNQV DAYAHRHLSI TGSQIWQNOK LPSANKLVTN 801 GVLALNARYS QIADNTTLRA GAINLTAGTA LVKRGNINWS TVSTKTLEDN 851 AELKPLAGRL NIEAGSGTLT IEPANRISAH TDLSIKTGGK LLLSAKGGNA 901 GAFSAQVSSL EAKGNIRLVT GETDLRGSKI TAGKNLVVAT TKGKLNIEAV 951 NNSFSNYFPT QKAAELNQKS KELEQQIAQL KKSSPKSKLI PTLQEERDRL 1001 AFYIQAINKE VKGKKPKGKE YLQAKLSAQN IDLISAQGIE ISGSDITASK 1051 KLNLHAAGVL PKAADSEAAA ILIDGITDQY EIGKPTYKSH YOKAALNKPS 1101 RLTGRTGVSI HAAAALDDAR IIIGASEIKA FSGSIDIKAH SDIVLEAGQN 1151 DAYTFLKTKG KSGKIIRXTK FTSTRDHLIM PAPVELTANG ITLQAGGNIE 1201 ANTTRFNAPA GKVTLVAGEE LQLLAEEGIH KHELDVQKSR RFIGIKVGKS 1251 NYSKNELNET KLPVRVVAQT AATRSGWDTV LEGTEFKTTL AGADIQAGVG 1301 EKAPADAKII LKGIVNRIQS EEKLETNSTV WQKQAGRGST IETLKLPSFE 1351 SPTPPKLTAP GGYIVDIPKG NLKTEIEKLA KQPEPEYLKQ LQVAKNVNWN 1401 QVQLAYDKWD YKQEGLTRAG AAIVTIIVTA LTYGYGATAA GGVAASGSST 1451 AAAAGTAATT TAAATTVSTA TANQTAALAS LYSQAAVSII NNKGDVGKAL 1501 KDLGTSDTVK QIVISALTAG ALNQMGADIA QLNSKVRTEL FSSTGVQTIA 1551 NLGGRLATNL SNAGISAGIN TAVNGGSLKD NLGNAALGAL VNSFQGEAAS 1601 KIKTTFSDDY VAKQFAHALA GCVSGLVQGK CKDGAIGAAV GEIVADSNLG 1651 GRNPATLSDA EKHKVISYSK IIAGSVAALN GGDVNTAANA AEVAVVNNAL 1701 NFDSTPTNAK KNQPQKPDKT ALEKIIQGIM PAHAAGAMTN PQDKDAAIWI 1751 SNIRNGITGP IVITSYGVYA AGWTAPLIGT AGKLAISTCM ANPSGCTVNV 1801 TQAAEAGAGI ATGAVTVGNA WEAPVGALSK AKAAKQAIPT QTVKELDGLL 1851 QESKNIGAVN TRINIANSTT RYTPNRQTGQ PVSAGFENVL EGHFHRPIAN 1901 NRSVFTXSPN ELKVILQSNK VVSSPVSMTP DGQYNRTVDV GKVIGTTSIK 1951 EGGQPTTTIK VFTDKSGNLI TTYPVKGN*

Computer analysis of this amino acid sequence predicts a transmembrane region and also gives the following results:

Homology with a Predicted ORF from N. meningitidis (Strain A)

ORF114 shows 91.9% identity over a 284 aa overlap with an ORF (ORF114a) from strain A of N. meningitidis:                           10        20        30        40 orf114.pep                   AVAETANSQGKGKQAGSSVSVSLKTSGDLCGKLKTTLKTLVC                   |||||||||||||||||||||||||||||||||||||||||| orf114a MNKGLHRIIFSKKHSTMVAVAETANSQGKGKQAGSSVSVSLKTSGDLCGKLKTTLKTLVC         10        20        30        40        50        60       50        60        70        80        90       100 orf114.pep SLVSLSMVLPAHAQITTDKSAPKNQQVVILKTNTGAPLVNIQTPNGRGLSHNRXYAFDVD |||||||      ||||||||||| ||||||||||||||||||||||||||||   |||| orf114a SLVSLSMXXXXXXQITTDKSAPKNXQVVILKTNTGAPLVNIQTPNGRGLSHNRYTQFDVD         70        80        90        100        110        120      110       120       130       140       150       160 orf114.pep NKGAVLNNDRNNNPFVVKGSAQLILNEVRGTALKLNGIVTVGGQKADVIIANPNGITVNG |||||||||||||||:|||||||||||||||||||||||||||||||||||||||||||| orf114a NKGAVLNNDRNNNPFLVKGSAQLILNEVRGTASKLNGIVTVGGQKADVIIANPNGITVNG        130       140       150       160       170       180      170       180       190       200       210       220 orf114.pep GGFKNVGRGILTTGAPQIGKDGALTGFDVVKAHWTVXAAGWNDKGGAXYTGVLARAVALQ |||||||||||| |||||||||||||||| ::  || |||||||||| |||||||||||| orf114a GGFKNVGRGILTIGAPQIGKDGALTGFDVRQGTLTVAAAGWNDKGGADYTGVLARAVALQ        190       200       210       220       230       240      230       240       250       260       270       290 orf114.pep GKXXGKXLAVSTGPQKVDYASGEISAGTAAGTKPTIALDTAALGGMYADSITLIANEKGV ||  || |||||||||||||||||||||||||||||||||||||||||||||||| |||| orf114a GKLQGKNLAVSTGPQKVDYASGEISAGTAAGTKPTIALDTAALGGMYADSITLIAXEKGV        250       260       270       280       290       300 orf114.pep GVX || orf114a GVKNAGTLEAAKQLIVTSSGRIENSGRIATTADGTEASPTYLXIETTEKGAXGTFISNGG        310       320       330       340       350       360

The complete length ORF114a nucleotide sequence <SEQ ID 61> is: 1 ATGAATAAAG GTTTACATCG CATTATCTTT AGTAAAAAGC ACAGCACCAT 51 GGTTGCAGTA GCCGAAACTG CCAACAGCCA GGGCAAAGGT AAACAGGCAG 101 GCAGTTCGGT TTCTGTTTCA CTGAAAACTT CAGGCGACCT TTGCGGCAAA 151 CTCAAAACCA CCCTTAAAAC CTTGGTCTGC TCTTTGGTTT CCCTGAGTAT 201 GGNATTNCNN NNCNNTNCCC AAATTACCAC CGACAAATCA GCACCTAAAA 251 ACCANCAGGT CGTTATCCTT AAAACCAACA CTGGTGCCCC CTTGGTGAAT 301 ATCCAAACTC CGAATGGACG CGGATTGAGC CACAACCGCT ATACGCAGTT 351 TGATGTTGAC AACAAAGGGG CAGTGTTAAA CAACGACCGT AACAATAATC 401 CGTTTCTGGT CAAAGGCAGT GCGCAATTGA TTTTGAACGA GGTACGCGGT 451 ACGGCTAGCA AACTCAACGG CATCGTTACC GTAGGCGGTC AAAAGGCCGA 501 CGTGATTATT GCCAACCCCA ACGGCATTAC TGTTAATGGC GGCGGCTTTA 551 AAAATGTCGG TCGGGGCATC TTAACTATCG GTGCGCCCCA AATCGGCAAA 601 GACGGTGCAC TGACAGGATT TGATGTGCGT CAAGGCACAT TGACCGTAGG 651 AGCAGCAGGT TGGAATGATA AAGGCGGAGC CGACTACACC GGGGTACTTG 701 CTCGTGCAGT TGCTTTGCAG GGGAAATTAC AAGGTAAAAA CCTGGCGGTT 751 TCTACCGGTC CTCAGAAAGT AGATTACGCC AGCGGCGAAA TCAGTGCAGG 801 TACGGCAGCG GGTACGAAAC CGACTATTGC CCTTGATACT GCCGCACTGG 951 GCGGTATGTA CGCCGAGAAA ATCACACTGA TTGCCAATGA AAAAGGCGTA 901 GGcGTCAAAA ATGCCGGCAC ACTCGAAGCG GCCAAGCAAT TGATTGTGAC 951 TTCGTCAGGC CGCATTGAAA ACAGCGGCCG CATCGCCACC ACTGCCGACG 1001 GCACCGAAGC TTCACCGACT TATCTNNCNA TCGAAACCAC CGAAAAAGGA 1051 GCNNCAGGCA CATTTATCTC CAATGGTGGT CGGATCGAGA GCAAAGGCTT 1101 ATTGGTTATT GAGACGGGAG AAGATATCAT CTTGCGTAAC GGAGCCGTGG 1151 TGCAGAATAA CGGCAGTCGC CCAGCTACCA CGGTATTAAA TGCTGGTCAT 1201 AATTTGGTGA TTGAGAGTAA AACTAATGTG AACAATGCCA AAGGCTCGNC 1251 TAATCTGTCG GCCGGCGGTC GTACTACGAT CAATGATGCT ACTATTCAAG 1301 CGGGCAGTTC CGTGTACAGC TCCACCAAAG GCGATACTGA NTTGGGTGAA 1351 AATACCCGTA TTATTGCTGA AAACGTAACC GTATTATCTA ACGGTAGTAT 1401 TGGCAGTGCT GCTGTAATTG AGGCTAAAGA CACTGCACAC ATTGAATCGG 1451 GCAAACCGCT TTCTTTAGAA ACCTCGACCG TTGCCTCCAA CATCCGTTTG 1501 AACAACGGTA ACATTAAAGG CGGAAAGCAG CTTGCTTTAC TGGCAGACGA 1551 TAACATTACT GCCAAAACTA CCAATCTGAA TACTCCCGGC AATCTGTATG 1601 TTCATACAGG TAAAGATCTG AATTTGAATG TTGATAAAGA TTTGTCTGCC 1651 GCCAGCATCC ATTTGAAATC GGATAACGCT GCCCATATTA CCGGCACCAG 1701 TAAAACCCTC ACTGCCTCAA AAGACATGGG TGTGGAGGCA GGCTTGCTGA 1751 ATGTTACCAA TACCAATCTG CGTACCAACT CGGGTAATCT GCACATTCAG 1801 GCAGCCAAAG GCAATATTCA GCTTCGCAAT ACCAAGCTGA ACGCAGCCAA 1851 GGCTCTCGAA ACCACCGCAT TGCAGGGCAA TATCGTTTCA GACGGCCTTC 1901 ATGCTGTTTC TGCAGACGGT CATGTATCCT TATTGGCCAA CGGTAATGCC 1951 GACTTTACCG GTCACAATAC CCTGACAGCC AAGGCCGATG TCNATGCAGG 2001 ATCGGTTGGT AAAGGCCGTC TGAAAGCAGA CAATACCAAT ATCACTTCAT 2051 CTTCAGGAGA TATTACGTTG GTTGCCGNNN NCGGTATTCA GCTTGGTGAC 2101 GGAAAACAAC GCAATTCAAT CAACGGAAAA CACATCAGCA TCAAAAACAA 2151 CGGTGGTAAT GCCGACTTAA AAAACCTTAA CGTCCATGCC AAAAGCGGGG 2201 CATTGAACAT TCATTCCGAC CGGGCATTGA GCATAGAAAA TACNAAGCTG 2251 GAGTCTACCC ATAATACGCA TCTTAATGCA CAACACGAGC GGGTAACGCT 2301 CAACCAAGTA GATGCCTACG CACACCGTCA TCTAAGCATT ANCGGCAGCC 2351 AGATTTGGCA AAACGACAAA CTGCCTTCTG CCAACAAGCT GGTGGCTAAC 2401 GGTGTATTGG CAATCAATGC GCGCTATTCC CAAATTGCCG ACAACACCAC 2451 GCTGAGAGCG GGTGCAATCA ACCTTACTGC CGGTACCGCC CTAGTCAAGC 2501 GCGGCAACAT CAATTGGATT ACCGTTTCGA CCAAGACTTT GGAAGATAAT 2551 GCCGAATTAA AACCATTGGC CGGACGGCTG AATATTGAAG CAGGTAGCGG 2601 CACATTAACC ATCGAACCTG CCAACCGCAT CAGTGCGCAT ACCGACCTGA 2651 GCATCAAAAC AGGCGGAAAA TTGCTGTTGT CTGCAAAAGG AGGAAATGCA 2701 GGTGCGCNTA GTGCTCAAGT TTCCTCATTG GAAGCAAAAG GCAATATCCG 2751 TCTGGTTACA GGAGNAACAG ATTTAAGAGG TTCTAAAATT ACAGCCGGTA 2901 AAAACTTGGT TGTCGCCACC ACCAAAGGCA AGTTGAATAT CGAAGCCGTA 2951 AACAACTCAT TCAGCAATTA TTTTCNTACA CAAAAAGNGN NNGNNCTCAA 2901 CCAAAAATCC AAAGAATTGG AACAACAGAT TGCGCAGTIG AAAAAAAGCT 2951 CGCNTAAAAG CAAGCTGATT CCAACCCTGC AAGAAGAACG CGACCGTCTC 3001 GCTTTCTATA TTCAAGCCAT CAACAAGGAA GTTAAAGGTA AAAAACCCAA 3051 AGGCAAAGAA TACCTGCAAG CCAAGCTTTC TGCACAAAAT ATTGACTTGA 3101 TTTCCGCACA AGGCATCGAA ATCAGCGGTT CCGATATTAC CGCTTCCAAA 3151 AAACTGAACC TTCACGCCGC AGGCGTATTG CCAAAGGCAG CAGATTCAGA 3201 GGCGGCTGCT ATTCTGATTG ACGGCATAAC CGACCAATAT GAAATTGGCA 3251 AGCCCACCTA CAAGAGTCAC TACGACAAAG CTGCTCTGAA CAAGCCTTCA 3301 CGTTTGACCG GACGTACGGG GGTAAGTATT CATGCAGCTG CGGCACTCGA 3351 TGATGCACGT ATTATTATCG GTGCATCCGA AATCAAAGCT CCCTCAGGCA 3401 GCATAGACAT CAAAGCCCAT AGTGATATTG TACTGGAGGC TGGACAAAAC 3451 GATGCCTATA CCTTCTTAAA AACCAAAGGT AAAAGCGGCA NAATNATCAG 3501 AAAAACNAAG TTTACCAGCA CCNGCGANCA CCTGATTATG CCAGCCCCNG 3551 TCGAGCTGAC CGCCAACGGT ATCACGCTTC ACGCAGGCGG CAACATCGAA 3601 GCTAATACCA CCCGCTTCAA TGCCCCTGCA GGTAAAGTIA CCCTGGTTGC 3651 GGGTGAANAG NTGCAACTGC TGGCAGAAGA AGGCATCCAC AAGCACGAGT 3701 TGGATGTCCA AAAAAGCCGC CGCTTTATCG GCATCAAGGT AGGTNAGAGC 3751 AATTACAGTA AAAACGAACT GAACGAAACC AAATTGCCTG TCCGCGTCGT 3801 CGCCCAAAAT GCAGCCACCC GTTCAGGCTG GGAThCCGTG CTCGAAGGTA 3851 CCGAATTCAA ATCCACGCTG GCCGGTGCCG ACATTCAGGC AGGTGTANGC 3901 GAAAAAGCCC GTGTCGATGC GAAAATCATC CTCAAAGGCA TTGTGAACCG 3951 TATCCAGTCG GAAGAAAAAT TAGAAACCAA CTCAACCGTA TGGCAGAAAC 4001 AGGCCGGACG CGGCAGCACT ATCGAAACGC TAAAACTGCC CAGCTTCGAA 4051 AGCCCTACTC CGCCCAAATT GTCCGCACCC GGCGGNTATA TCGTCGACAT 4101 TCCGAAAGGC AATCTGAAAA CCGAAATCGA AAAGCTGTCC AAACAGCCCG 4151 AGTATGCCtA TCTGAAACAG CTCCAAGTAG CGAAAAACAT CAACTGGAAT 4201 CAGGTGCAGC TTGCTTACGA CAGATGGGAC TACAAACAGG AGGGCTTAAC 4251 CGAAGCAGGT GCGGCGATTA TCGCACTGGC CGTTACCGTG GTCACCTCAG 4301 GCGCAGGAAC CGGAGCCGTA TTGGGATTAA ACGGTGCGNC CGCCGCCGCA 4351 ACCGATGCAG CATTCGCCTC TTTGGCCAGC CAGGCTTCCG TATCGTTCAT 4401 CAACAACAAA GGCGATGTCG GCAAAACCCT GAAAGAGCTG GGCAGAAGCA 4451 GCACGGTGAA AAATCTGGTG GTTGCCGCCG CTACCGCAGG CGTAGCCGAC 4501 AAAATCGGCG CTTCGGCACT GANCAATGTC AGCGATAAGC AGTGGATCAA 4551 CAACCTGACC GTCAACCTAG CCAATGNCGG GCAGTGCCGC ACTGAttaa

This encodes a protein having amino acid sequence <SEQ ID 62>: 1 MNKGLHRIIF SKKHSTMVAV AETANSQGKG KQAGSSVSVS LKTSGDLCGK 51 LKTTLKTLVC SLVSLSMXXX XXXQITTDKS APIDXQVVIL KTNTGAPLVN 101 IQTPNGRGLS HNRYTQFDVD NKGAVLNNDR NNNPFLVKGS AQLILNEVRG 151 TASKLNGIVT VGGQKADVII ANPNGITVNG GGFKNVGRGI LTIGAPQIGK 201 DGALTGFDVR QGTLTVGAAG WNDKGGADYT GVLARAVALQ GKLQGKNLAV 251 STGPQKVDYA SGEISAGTAA GTKPTIALDT AALGGMYADS ITLTAXEKGV 301 GVKNAGTLEA AKQLIVTSSG RIENSGRIAT TADGTEASPT YLXIETTEKG 351 AXGTFISNGG RIESKGLLVI ETGEDIXLPA GAVVQNNGSR PATTVLNAGH 401 NLVIESKTNV NNAXGSXNLS AGGRTTINDA TIQAGSSVYS STKGDTXLGE 451 NTRIIAENVT VLSNGSIGSA AVIEAKDTAN IESGKPLSLE TSTVASNIRL 501 NNGNIKGGKQ LALLADDNIT AKTTNLNTPG NLYVHTGKDL NLNVDKDLSA 551 ASIHLKSDNA AHITGTSKTL TASKDNGVEA GLLNVTNTNL RTNSGNLHIQ 601 AAKGNZQLRH TKLNAAKALE TTALQGNIVS DGLHAVSADG HVSLLANGNA 651 DFTGHNTLTA KADVXAGSVG KGRLKADNTN ITSSSGDITL VAXXGIQLGD 701 GKQRNSINGK HISIKNNGGN ADLKNLNVHA KSGALNIHSO RALSIENTKL 751 ESTHNTHLNA QHERVTLNQV DAYAHRHLSI XGSQIWQNDK LPSANKLVAN 801 GVLAXNARYS QIADNTTLRA CAINLTAGTA LVKRGNINWS TVSTKTLEDN 851 AELKPLAGRL NIEAGSGTLT IEFANRISAH TDLSIKTGGK LLLSAXGGNA 901 GAXSAQVSSL EAKGNIRLVT GXTDLRGSKI TAGKNLVVAT TKGKLNIEAV 951 NNSFSNYFXT QKXXXLNQKS KELEOQIAQL KKSSXKSKLI PTLQEERDRL 1001 AFYIQAINKE VKGKKPKGKE YLQAXLSAQN IDLISAQGIE ISGSDITASK 1051 KLNLHAAGVL PKAADSEAAA ILIDGITOQY EIGKPTYKSH YDKAALNKPS 1101 RLTGRTGVSI HAAAALDDAR IIIGASEIKA PSGSIDIKAR SDIVLEAGQN 1151 DAYTFLXTKG KSGXXIRKTK FTSTXXHLIM PAPVELTANG ITLQAGGNIE 1201 ANTTRFHAPA GKVTLVAGEK XQLLAEEGIK KHELDVQKSR RFIGIKVGXS 1251 NYSINELNET KLPVRVVAQX AATRSGWDTV LEGTEFKTTL AGADIQAGVX 1301 EKARVQAXII LKGIVNRIQS EEKLETNSTV WQKQAGRGST IETLKLPSFE 1351 SPTPPKLSAP GGYIVDIPKG NLKTEIEKLS KQPEYAYLKQ LQVAKNINWN 1401 QVQLAYQRWD YKQEGLTEAG AAIIALAVTV VTSGAGTGAV LGLNGAXAAA 1451 TDAAFASLAS QASVSFINNK GDVGKTLKEL GRSSTVKNLV VAAATAGVAD 1501 KIGASALXNV SDKQWINNLT VNLANXGQCR TD*

ORF114-1 and ORF114a show 89.8% identity in 1564 aa overlap orf114a.pep MNKGLHRIIFSKKHSTMVAVAETANSQGKGKQAGSSVSVSLKTSGDLCGKLKTTLKTLVC |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| orf114-1 MNKGLHRIIFSKKHSTMVAVAETANSQGKGKQAGSSVSVSLKTSGDLCGKLKTTLKTLVC orf114a.pep SLVSLSMXXXXXXQITTDKSAPKNXQVVILKTNTGAPLVNIQTPNGRGLSHNRYTQFDVD |||||||      ||||||||||| ||||||||||||||||||||||||||||||||||| orf114-1 SLVSLSMXXXXXXQITTDKSAPKNQQVVILKTNTGAPLVNIQTPNGRGLSHNRYTQFDVD orf114a.pep NKGAVLNNDRNNNPFLVKGSAQLILNEVRGTASKLNGIVTVGGQKADVIIANPNGITVNG |||||||||||||||:|||||||||||||||||||||||||||||||||||||||||||| orf114-1 NKGAVLNNDRNNNPFVVKGSAQLILNEVRGTASKLNGIVTVGGQKADVIIANPNGITVNG orf114a.pep GGFKNVGRGILTIGAPQIGKDGALTGFDVRQGTLTVGAAGWNDKGGADYTGVLARAVALQ |||||||||||| ||||||||||||||||||||||||||||||||||||||||||||||| orf114-1 GGFKNVGRGILTTGAPQIGKDGALTGFDVRQGTLTVGAAGWNDKGGADYTGVLARAVALQ orf114a.pep GKLQGKNLAVSTGFQKVDYASGEISAGTAAGTKPTIALDTAALGGMYADSITLIAXEKGV ||||||||||||||||||||||||||||||||||||||||||||||||||||||| |||| orf114-1 GKLOGKNLAVSTGPQKVDYASGEISAGTAAGTKPTIALDTAALGGMYADSITLIANEKGV orf114a.pep GVKNAGTLEAAXQLIVTSSGRIENSGRIATTADGTLASPTYLXIETTEKGAXGTFISNGG |||||||||||||||||||||||||||||||||||||||||| |||||||| |||||||| orf114-1 GVKNAGTLEAAXQLIVTSSGRIENSGRIATTADGTLASPTYLSIETTEKGAAGTFISNGG orf114a.pep RIESKGLLVIETGEDIXLRNGAVVQNNGSRPATTVLNAGHNLVIESKTNVNNAKGSXNLS |||||||||||||||| ||||||||||||||||||||||||||||||||||||||  :|| orf114-1 RIESKGLLVIETGEDISLRNGAVVQNNGSRPATTVLNAGHNLVIESKTNVNNAKGPANLS orf114a.pep AGGRTTINDATIQAGSSVYSSTKGDTXLGENTRIIAENVTVLSNGSIGSAAVIEAKDTAH | |||:|::|:||:|::||||:||:: ||:|||| : :|||||||||:|:|||:|||||| orf114-1 ADGRTVIKEASIQTGTTVYSSSKGNAELGNNTRITGADVTVLSNGTISSSAVIDAKDTAH orf114a.pep IESGKPLSLETSTVASNIRLNNGNIKGGKQLALLADDNITAKTTNLNTPGNLYVHTGKDL ||:|||||||||||:|||:|:||||:|:|||||||||||||||||||||||||||||||| orf114-1 IEAGKPLSLEASTVTSDIRLNGGSIKGGKQLALLADDNITAKTTNLNTPGNLYVHTGKDL orf114a.pep NLNVDKDLSAASIHLKSDNAAHITGTSKTLTASKDMGVEAGLLNVTNTNLRTNSGNLHIQ ||||||||||||||||||||||||||||||||||||||||| |||||||||||||||||| orf114-1 NLNVDKDLSAASIHLKSDNAAHITGTSKTLTASKDMGVEAGSLNVTNTNLRTNSGNLHIQ orf114a.pep AAKGNIQLRNTKLNAAKALETTALQGNIVSDGLHAVSADGHVSLLANGNADFTGHNTLTA |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| orf114-1 AAKGNIQLRNTKLNAAKALETTALQGNIVSDGLHAVSADGHVSLLANGNADFTGHNTLTA orf114a.pep KADVXAGSVGKGRLKADNTNITSSSGDITLVAXXGIQLGDGKQRNSINGKHISIKNNGGN |||| |||||||||||||||||||||||||||  |||||||||||||||||||||||||| orf114-1 KADVNAGSVGKGRLKADNTNITSSSGDITLVAGNGIQLGDGKQRNSINGKHISIKNNGGN orf114a.pep ADLKNLNVHAKSGALNIHSDRALSIENTKLESTHNTHLNAQHERVTLNQVDAYAHRHLSI |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| orf114-1 ADLKNLNVHAKSGALNIHSDRALSIENTKLESTHNTHLNAQHERVTLNQVDAYAHRHLSI orf114a.pep XGSQIWQNDKLPSANKLVANGVLAXNARYSQIADNTTLRAGAINLTAGTALVKRGNINWS :||||||||||||||||||||||| ||||||||||||||||||||||||||||||||||| orf114-1 TGSQIWQNDKLPSANKLVANGVLALNARYSQIADNTTLRAGAINLTAGTALVKRGNINWS orf114a.pep TVSTKTLEDNAELKPLAGRLNIEAGSGTLTIEPANRISAHTDLSIKTGGKLLLSAKGGNA |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| orf114-1 TVSTKTLEDNAELKPLAGRLNIEAGSGTLTIEPANRISAHTDLSIKTGGKLLLSAKGGNA orf114a.pep GAXSAQVSSLEAKGNIRLVTGXTDLRGSKITAGKNLVVATTKGKLNIEAVNNSFSNYFXT || |||||||||||||||||| |||||||||||||||||||||||||||||||||||| | orf114-1 GAPSAQVSSLEAKGNIRLVTGETDLRGSKITAGKNLVVATTKGKLNIEAVNNSFSNYFPT orf114a.pep QKXXXLNQKSKELEQQIAQLKKSSXKSKLIPTLQEERDRLAFYIQAINKEVKGKKPKGKE ||   ||||||||||||||||||| ||||||||||||||||||||||||||||||||||| orf114-1 QKAAELNQKSKELEQQIAQLKKSSPKSKLIPTLQEERDRLAFYIQAINKEVKGKKPKGKE orf114a.pep YLQAKLSAQNIDLISAQGIEISGSDITASKKLNLHAAGVLPKAADSEAAAILIDGITDQY |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| orf114-1 YLQAKLSAQNIDLISAQGIEISGSDITASKKLNLHAAGVLPKAADSEAAAILIDGITDQY orf114a.pep EIGKPTYKSHYDKAALNKPSRLTGRTGVSIHAAAALDDARIIIGASEIKAPSGSIDIKAH |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| orf114-1 EIGKPTYKSHYDKAALNKPSRLTGRTGVSIHAAAALDDARIIIGASEIKAPSGSIDIKAH orf114a.pep SDIVLEAGQNDAYTFLXTKGKSGXXIRKTKFTSTXXHLIMPAPVELTANGITLQAGGNIE |||||||||||||||| ||||||  |||||||||  |||||||||||||||||||||||| orf114-1 SDIVLEAGQNDAYTFLKTKGKSGKIIRKTKFTSTRDHLIMPAPVELTANGITLQAGGNIE orf114a.pep ANTTRFNAPAGKVTLVAGEXXQLLAEEGIHKHELDVQKSRRFIGIKVGXSNYSKNELNET |||||||||||||||||||  ||||||||||||||||||||||||||| ||||||||||| orf114-1 ANTTRFNAPAGKVTLVAGEELQLLAEEGIHKHELDVQKSRRFIGIKVGKSNYSKNELNET orf114a.pep KLPVRVVAQXAATRSGWDTVLEGTEFKTTLAGADIQAGVXEKARVDAKIILKGIVNRIQS |||||||||:||||||||||||||||||||||||||||| ||||:||||||||||||||| orf114-1 KLPVRVVAQTAATRSGWDTVLEGTEFKTTLAGADIQAGVGEKARADAKIILKGIVNRIQS orf114a.pep EEKLETNSTVWQKQAGRGSTIETLKLPSFESPTPPKLSAPGGYIVDIPKGNLKTEIEKLS ||||||||||||||||||||||||||||||||||||||:||||||||||||||||||||: orf114-1 EEKLETNSTVWQKQAGRGSTIETLKLPSFESPTPPKLSTPGGYIVDIPKGNLKTEIEKLA orf114a.pep KQPEYAYLKQLQVAKNINWNQVQLAYDRWDYKQEGLTEAGAAIIALAVTVVTSGAGTGAV ||||||||||||||||:||||||||||:|||||||||:|||||::: ||::| | |: |: orf114-1 KQPEYAYLKQLQVAKNVNWNQVQLAYDKWDYKQEGLTRAGAAIVTIIVTALTYGYGATAA orf114a.pep LGLNGA--------------XAATD----------AAFASLASQASVSFINNKGDVGKTL 1477  |: ::              :||||          ||:||| |||:||:|||||||||:| orf114-1 GGVAASGSSTAAAAGTAATTTAAATTVSTATAMQTAALASLYSQAAVSIINNKGDVGKAL 1500 orf114a.pep KELGRSSTVKNLVVAAATAGVADKIGA----------SALXNVSDKQWINNL----TVNL 1523 |:|| |:|||::|::| |||: :::||          : | : : :| | ||    ::|| orf114-1 KDLGTSDTVKQIVTSALTAGALNQMGADIAQLNSKVRTELFSSTGNQTIANLGGRLATNL 1560 orf114a.pep ANXGQCRTDX :| | orf114-1 SNAGISAGINTAVN... Homology with pspA Putative Secreted Protein of N. meningitidis (Accession Number AF030941)

ORF114 and pspA protein show 36% aa identity in 302 aa overlap: Orf114: 1 AVAETANSQGKGKQAGSSVSVSL----KTSGDXXXXXXXXXXXXXXXXXXXXXXXPAHAQ 56 AVAE  +  GK  Q   + SV +      S                         PA A pspA: 19 AVAENVHRDGKSMQDSEAASVRVTGAASVSSARAAFGFRMAAFSVMLALGVAAFSPAPAS 78 Orf114: 57 -ITTDKSAPKNQQVVILKTNTGAPLVNIQTPNGRGLSHNRXYAFDVDNKGAVLNNDRNN- 114  I  DKSAPKNQQ VIL+T  G P VNIQTP+ +G+S NR   FDVD KG +LNN R+N pspA: 79 GIIADKSAPKNQQAVILQTANGLPQVNIQTPSSQGVSVNRFKQFDVDEKGVILNNSRSNT 138 Orf114: 115 ----------NPFVVKGSAQLILNEV-RGTASKLNGIVTVGGQKADVIIANPNGITVNGG 163           NP + +G A++I+N++     S LNG + VGG++A+V++ANP+GI VNGG pspA: 139 QTQLGGWIQGNPHLARGEARVIVNQIDSSNPSLLNGYIEVGGKRAEVVVANPSGIRVNGG 198 Orf114: 164 GFKNVGRGILTTGAPQIGKDGALTGFDVVKAHWTVXAAGWNDKGGAXYTGVLARAVALQG 223 G  N     LT+G P +  +G LTGFDV      +   G  D   A YT +L+RA  + papA: 199 GLINAASVTLTSGVPVL-NNGNLTGFDVSSGKVVIGGKGL-DTSDADYTRILSRAAEINA 256 Orf114: 224 KXXGKXLAVSTGPQKVDYASGEISAGTAAGTK----PTIALDTAALGGMYADSITLIANE 279    GK + V +G  K+D+        +A  +     PT+A+DTA LGGMYAD ITLI+ + pspA: 257 GVWGKDVKVVSGKNKLDFDGSLAKTASAPSSSDSVTPTVAIDTATLGGMYAQKITLISTD 316 Orf114: 280 KG 291  G papA: 317 NG 318

ORF114a is also homologous to pspA: gil2623258 (AF030941) putative secreted protein (Neisseria meningitidis) Length = 2273 Score +32 261 bits (659), Expect +32 3e−69 Identities = 203/663 (30%), Positives 314/663 (46%), Gaps 76/663 (11%) Query:    1 MNKGLHRIIFSKKHSTMVAVAETANSQGKGKQAGSSVSVSLK-----TSGDXXXXXXXXX 55 MNK  +++IF+KK S M+AVAE  +  GK  Q   + SV +      +S Sbjct:    1 MNKRCYKVIFNKKRSCMMAVAENVHRDGKSMQDSEAASVRVTGAASVSSARAAFGFRMAA 60 Query:   56 XXXXXXXXXXXXXXXXXXQITTKDSAPKNXQVVILKTNTGAPLVNIQTPNGRGLSHNRYT 115                    I  DKSAPKN Q VIL+T  G P VNIQTP+ +G+S NR+ Sbjct:   61 FSVMLALGVAAFSPAPASGIIADKSAPKNQQAVILQTANGLPQVNIQTPSSQGVSVNRFK 120 Query:  116 QFDVDNKGAVLNNDRNN-----------NPFLVKGSAQLILNEV-RGTASKLNGIVTVGG 163 QFDVD KG +LNN R+N-----------NP L +G A++I+N++     S LNG + VGG Sbjct:  121 QFDVDEKGVILNNSRSNTQTQLGGWIQGNPHLARGEARVIVNQIDSSNPSLLNGYIEVGG 180 Query:  164 QKADVIIANPNGITVNGGGFKNVGRGILTIGAPQIGKDGALTGFDVRQGTLTVGAAGWND 223 ++A+V++ANP+GI VNGGG  N     LT G P +  +G LTGFDV  G + +G  G  D Sbjct:  181 KRAEVVVANPSGIRVNGGGLINAASVTLTSGVPVL-NNGNLTGFDVSSGKVVIGGKGL-D 238 Query:  224 KGGADYTGVLARAVALQGKLQGKNLAVSTGPQKVDYASGEISAGTAAGTK----PTIALD 279    ADYT +L+RA  +   + GK++ V +G  K+D+        +A  +     PT+A+D Sbjct:  239 TSDADYTRILSRAAEINAGVWGKDVKVVSGKNKLDFDGSLAKTASAPSSSDSVTPTVAID 298 Query:  280 TAALGGMYADSITLIAXEKGVGVKNAGTLEAAK-QLIVTSSGRIENSGRIATTADGTEAS 338 TA LGGMYAD ITLI+ + G  ++N G + AA   + +++ G++ NSG I Sbjct:  299 TATLGGMYADKITLISTDNGAVIRNKGRIFAATGGVTLSADGKLSNSGSI-------DAA 351 Query:  339 PTYLXIETTEKGAXGTFISNGGRIESKGLLVIETGEDIXLRNGAVVQNNGSRPATTVLNA 398    +  +T +        +  G I S    V++  + I  + G +    GS     + + Sbjct:  352 EITISAQTVD--------NRQGFIRSGKGSVLKVSDGINNQAGLI----GSAGLLDIRDT 399 Query:  399 GHNLVIESKTNVNNAKGS----XNLSAGGRTTINDATIQAGSSVYSSTKGDTXLGENTRI 454 G     +S  ++NN  G+     ++S   ++  ND  + A   V S +  D   G+ Sbjct:  400 G-----KSSLHINNTDGTIIAGKDVSLQAKSLDNDGILTAARDV-SVSLHDDFAGKRDIE 453 Query:  455 IAENVTVLSNGSIGSAAVIEAKDTAHIESGKPLSLETSTVASNIRLNNGNIKGGKQLALL 514     +T  + G + +  +I+A DT  + + +  +  +  + S  R       G     L+ Sbjct:  454 AGRTLTFSTQGRLKNTRIIQAGDTVSLTAAQIDNTVSGKIQSGNRTGLNGKNGITNRGLI 513 Query:  515 ADDNIT-----AKTTNLNTPGNLYVHTGKDLNLNVDKDLSAASIHLKSDNAAHITGTSKT 569   + IT     AK+ N  T G +Y   G  + +  D  L+          AA Sbjct:  514 NSNGITLLQTEAKSDNAGT-GRIY---GSRVAVEADTLLNREETVNGETKAA-------V 562 Query:  570 LTASKDMGVEAGXXXXXXXXXXXXSGNLHIQAA---KGNIQLRNTKL-NAAKALETTALQ 625 + A + + + A             SG+LHI +A      +Q  NT L N + A+E++ Sbjct:  563 IAARERLDIGAREIENREAALLSSSGDLHIGSALNGSRQVQGANTSLHNRSAAIESS--- 619 Query:  626 GNI 628 GNI Sbjct:  620 GNI 622 Score +32 37.5 bits (65), Expect = 0.53 Identities = 87/432 (20%), Positives +32 159/432 (36%), Gaps = 62/432 (14%) Query:  239 LQGKLQGKNLAVSTGPQKVDYASGEISAGTAAGTKPTIALDTAALGGMYADSITLIAXEK 298 LQG LQGKN+  + G    +  +G I A  A   K        A   + + S T     + Sbjct: 1023 LQGDLQGKNIFAAAGSDITN--TGSIGAENALLLK--------ASNNIESRSETRSNQNE 1072 Query:  299 GVGVKNAGTLEAAKQLIVTSSGRI--ENSGRIATTADGTEASPTYLXIETTEKGAXG-TF 355    V+N G + A   L    +G +  +    I  TA            E T +   G T Sbjct: 1073 QGSVRNIGRV-AGIYLTGRQNGSVLLDAGNNIVLTAS-----------ELTNQSEDGQTV 1120 Query:  356 ISNGGRIESKGLLVIETGEDIXLRNGAVVQNNGSRPATTVLNAGHNLVIESK-------T 408 ++ GG I S    +      I   +  V++   +   +T+   G NL + +K Sbjct: 1121 LNAGGDIRSDTTGISRNQNTIFDSDNYVIRKEQNEVGSTIRTRG-NLSLNAKGDIRIPAA 1179 Query:  409 NVNNAKGSXNLSAGGRTTINDATIQAGSS--------VYSSTKGDTXLGENTRIIAENVT 460  V + +G   L+AG      D  ++AG +         Y+   G     + TR + Sbjct: 1180 EVGSEQGRLKLAAG-----RDIKVEAGKAHTETEDALKYTGRSGGGIKQKMTRHLKNQNG 1234 Query:  461 VLSNGSIGSAAVIEAKDTAHIESGKPLSLETSTVASWIRLNNGNIKGGKQLALLADDNIT 520    +G++    +I         +G  +  +  T+ S    NN  +K  +  +  A+ N Sbjct: 1235 QAVSGTLDGKEIILVSGRDITVTGSNIIADNHTILS--AKNNIVLKAAETRSRSAEMNKK 1292 Query:  521 AKTTNLNTPG-NLYVHTGKDLNLNVDKDLSAASIHLKSDN-------AAHITGTSKTLTA 572  K+  + + G      + KD   N  + +S     + S N         H T T  T+++ Sbjct: 1293 EKSGLMGSGGIGFTAGSKKDTQTNRSETVSHTESVVGSLNGNTLISAGKHYTQTGSTISS 1352 Query:  573 SK-DMGVEAGXXXXXXXXXXXXSGNLHIQAAKG-----NIQLRNTKLNAAKALETTALQG 626  + D+G+ +G              +  +   KG     ++ + NT + A  A++     G Sbjct: 1353 PQGDVGISSGKISIDAAQNRYSQESKQVYEQKGVTVAISVPVVNTVMGAVDAVKAVQTVG 1412 Query:  627 NIVSDGLHAVSA 638    +  ++A++A Sbjct: 1413 KSKNSRVNAMAA 1424

Amino acids 1-1423 of ORF114-1 were cloned in the pGex vector and expressed in E. coli, as described above. GST-fusion expression was visible using SDS-PAGE, and FIG. 5 shows plots of hydrophilicity, antigenic index, and AMPHI regions for ORF114-1.

Based on these results, including the homology with the putative secreted protein of N. meningitidis and on the presence of a transmembrane domain, it is predicted that this protein from N. meningitidis, and its epitopes, could be useful antigens for vaccines or diagnostics.

Example 14

The following partial DNA sequence was identified in N. meningitidis <SEQ ID 63> 1 CGCTTCATTC ATGATGAAGC AGTCGGCAGC AACATCGGCG GCGGCAAAAT 51 GATTGTTGCA GCCGGGCAGG ATATCAATGT ACGCGGCAnA AGCCTTATTT 101 CTGATAAGGG CATTGTTTTA AAAGCAGGAC ACGACATCGA TATTTCTACT 151 GCCCATAATC GCTATACCGG CAATGAATAC CACGAGAGCA wAAAwTCAGG 201 CGTCATGGGT ACTGGCGGAT TGGGCTTTAC TATCGGTAAC CGGAAAACTA 251 CCGATGACAC TGATCGTACC AATATTGTsC ATACAGGCAG CATTATAGGC 301 AGCCTGAaTG GAGACACCGT TACAGTTGCA GGAAACCGCT ACCGACAAAC 351 CGGCAGTACC GTCTCCAGCC CCGACGGGCG CAATACCGTC ACAGCCAAAw 401 GCATAGATGT AGAGTTCGCA AACAACCGGT ATGCCACTGA CTACGcCCAT 451 ACCCAGGGAA CAAAAAGGCC TTACCGTCGC CCTCAATGTC CCGGTTGTCC 501 AAGCTGCACA AAACTTCATA CAAGCAGCCC AAAATGTGGG CAAAAGTAAA 551 AATAAACGCG TTAATGCCAT GGCTGCAGCC AATGCTGCAT GGCAGAGTTA 601 TCAAGCAACC CAACAAATGC AACAATTTGC TCCAAGCAGC AGTGCGGGAC 651 AAGGTCAAAA CTACAATCAA AGCCCCAGTA TCAGTGTGTC CATTAC.TAC 701 GGCGAACAGA AAAGTCGTAA CGAGCAAAAA AGACATTACA CCGAAgCGGC 751 AgCAAGTCAA ATTATCGGCA AAGGGCAAAC CACACTTGCG GCAACAGGAA 801 GTGGGGAGCA GTCCAATATC AATATTACAG GTTCCGATGT CATCGGCCAT 951 GCAGGTACTC C.CTCATTGC AAGCAACCAT ATCAGACTCC AATCTGCCAA 901 ACAGGACGGC AGCGAGCAAA GCAAAAACAA AAGCAGTGGT TGGAATGCAG 951 GCGTACGTnn CAAAATAGGC AAcGGCATCA GGTTTGGAAT TACCGCCGGA 1001 GGAAATATCG GTAAAGGTAA AGAGCAAGGG GGAAGTACTA CCCACCGCCA 1051 CACCCATGTC GGCAGCACAA CCGGCAAAAC TACCATCCGA AGCGGCGGGG 1101 GATACCACCC TCAAAGGTGT GCAGCTCATC GGCAAAGGCA TACAGGCAGA 1151 TACGCGCAAC CTGCATATAG AAAGTGTTCA AGATACTGAA ACCTATCAGA 1201 GCAAACAGCA AAACGGCAAT GTCCAAGTTt ACTGTCGGTT ACGGATTCAG 1251 TGCAAGCGGC AGTTACCGCC AAAGCAAAGT CAAAGCAGAC CATGCCTCCG 1301 TAACCGGGCA AAgCGGTATT TATGCCGGAG AAGACGGCTA TCAAATyAAA 1351 GTyAGAGACA ACACAGACCT yAAGGGCGGT ATCATCACGT CTAGCCAAAG 1401 CGCAGAAGAT AAGGGCAAAA ACCTTTTTCA GACGGCCACC CTTACTGCCA 1451 GCGACATTCA AAACCACAGC CGCTACGAAG GCAGAAGCTT CGGCATAGGC 1501 GGCAGTTTCG ACCTGAACGG CGGCTGGGAC GGCACGGTTA CCGACAAACA 1551 AGGCAGGCCT ACCGACAGGA TAAGCCCGGC AGCCGGCTAC GGCAGCGACG 1601 GAGACAGCAA AAACAGCACC ACCCGCAGCG GCGTCAACAC CCACAACATA 1651 CACATCACCG ACGAAGCGGG ACAACTTGCC CGAACAGGCA GGACTGCAAA 1701 AGAAACCGAA GCGCGTATCT ACACCGGCAT CGACACCGAA ACTGCGGATC 1751 AACACTCAGG CCATCTGAAA AACAGCTTCG AC...

This corresponds to the amino acid sequence <SEQ ID 64; ORF116>: 1 ..RFIHDEAVGS NIGGGKNIVA AGQDINVRGX SLISDKGIVL KAGADIDIST 51   AHNRYTGNEY HESXXSGVMG TGGLGFTIGN RKTTDDTDRT NIVHTGSIIG 101   SLNGDTVTVA GNRYRQTGST VSSPEGRNTV TAKXIDVEFA NNRYATDYAH 151   TQEQKGLTVA LNVPVVQAAQ NFIQAAQNVG KSKNKRVNAM AAANAAWQSY 201   QATQQMQQFA PSSSAGQGQN YNQSPSISVS IXYGEQKSRN EQKRNYTEAA 251   ASQIIGKGQT TLAATGSGEQ SNINITGSDV IGHAGTXLIA DNHIRLQSAX 301   QDGSEQSKNK SSGWNAGVRX KIGNGIRFGI TAGGNIGKGK EQGGSTTHRH 351   THVGSTTGKT TIRSGGDTTL KGVQLIGXGI QADTRNLHIE SVQDTETYQS 401   KQQNGNVQVT VGYGFSASGS YRQSKVKADH ASVTGQSGIY AGEDGYQIKV 451   RDNTDLKGGI ITSSQSAEDK GKNLFQTATL TASDIQNHSR YEGRSFGIGG 501   SFDLNGGWDG TVTDKQGRPT DRISPAAGYG SDGDSKNSTT RSGVNTHNIH 551   ITDEAGQLAR TGRTAKETEA RIYTGIDTET ADQHSGHLKN SFD...

Computer analysis of this amino acid sequence gave the following results:

Homology with pspA Putative Secreted Protein of N. meningitidis (Accession Number AF030941)

ORF116 and pspA protein show 38% aa identity in 502 aa overlap: Orf116: 6    EAVGSNIGGGKMIVAAGQDINVRGXSLISDKGIVLKAGHDIDISTAHNRYTGNEYHESXX 65      +AV   + G ++I+ +G+DI V G ++I+D   +L A ++I +  A  R    E ++ PspA: 1235 QAVSGTLDGKEIILVSGRDITVTGSNIIADNHTILSAKNNIVLKAAETRSRSAEMNKKEK 1294 Orf116: 66   XXXXXXXXXXXXXXNRKXXXXXXRTNIVHTGSIIGSLNGDTVTVAGNRYRQTGSTVSSPE 125                    ++K         + HT S++GSLNG+T+  AG  Y QTGST+SSP+ PspA: 1295 SGLMGSGGIGFTAGSKKDTQTNRSETVSHTESVVGSLNGNTLISAGKHYTQTGSTISSPQ 1354 Orf116: 126  GRNTVTAKXIDVEFANNRYATDYAHTQEQKGLTVALNVPXXXX---XXXXXXXXXXXGKS 182      G   +++  I ++ A NRY+ +     EQKG+TVA++VP               GKS PspA: 1355 GDVGISSGKISIDAAQNRYSQESKQVYEQKGVTVAISVPVVNTVMGAVDAVKAVQTVGKS 1414 Orf116: 183  KNKRVXXXXXXXXXWQSYQATQQMQQFA--PSSSAGQGQNYNQSPSISVSIXYGEQKSRN 240      KN RV          +   +   +   A  P  +AGQG        ISVS+ YGEQK+ + PspA: 1415 KNSRVNAMAAANALNKGVDSGVALYNAARNPKKAAGQG--------ISVSVTYGEQKNTS 1466 Orf116: 241  EQKRHYTEAAASQIIGKGQTTLAATGSGEQSNINITGSDVIGHAGTXLIADNHIRLQSAK 300      E +   T+    +I G G+ +L A+G+G+ S I ITGSDV G  GT L A+N +++++A+ PspA: 1467 ESRIKGTQVQEGKITGGGKVSLTASGAGKDSRITITGSDVYGGKGTRLKAENAVQIEAAR 1526 Orf116: 301  QDGSEQSKNKSSGWNAGVRXKIGNGIRFGITAXXXXXXXXXXXXSTTHRHTHVGSTTGKT 360      Q   E+S+NKS+G+NAGV   I  GI FG TA             T +R++H+GS   +T PspA: 1527 QTHQERSENKSAGFNAGVAIAINKGISFGFTAGANYGKGYGNGDETAYRNSHIGSKDSQT 1586 Orf116: 361  TIRSGGDTTLKGVQLIGKGIQADTRNLHIESVQDTETYQSKQQNGNVQVTVGYGFSASGS 420       I SGGDT +KG QL GKG+     +LHIES+QDT  ++ KQ+N + QVTVGYGFS  GS PspA: 1587 AIESGGDTVIKGGQLKGKGVGVTAESLHIESLQDTAVFKGKQENVSAQVTVGYGFSVGGS 1646 Orf116: 421  YRQSKVKADHASVTGQSGIYAGEDGYQIKVRDNTDLKGGIITSSQSAEDKGKNLFQTATL 480      Y +SK  +D+ASV  QSGI+AG DGY+I+V   T L G  + S     DK KNL +T+ + PspA: 1647 YNRSKSSSDYASVNEQSGIFAGGDGYRIRVNGKTGLVGAAVVSD---ADKSKNLLKTSEI 1703 Orf116: 481  TASDIQNHSRYEGRSFGIGGSF 502         DIQNH+     + G+ G F PspA: 1704 WHKDIQNHASAAASALGLSGGF 1725

Based on homology with pspA, it is predicted that this protein from N. meningitidis, and its epitopes, could be useful antigens for vaccines or diagnostics.

Example 15

The following partial DNA sequence was identified in N. meningitidis SEQ ID 65> 1 ..ACGACCGGCA GCCTCGGCGG CATACTGGCC GGCGGCGGCA CTTCCCTTGC 51   CGCACCGTAT TTGGACAAAG CGGCGGAAAA CCTCGGTCCG GCGGGCAAAG 101   CGGCGGTCAA CGCACTGGGC GGTGCGGCCA TCGGCTATGC AACTGGTGGT 151   AGTGGTGGTG CTGTGGTGGG TGCGAATGTA GATTGGAACA ATAGGCAGCT 201   GCATCCGAAA GAAATGGCGT TGGCCGACAA ATATGCCGAA GCCCTCAAGC 251   GCGAAGTTGA AAAACGCGAA GGCAGAAAAA TCAGCAGCCA AGAAGCGGCA 301   ATGAGAATCC GCAGGCAGAT ATGCGTTGGG TGGACAAAGG TTCCCAAGAC 351   GGCTATACCG ACCAAAGCGT CATATCCCTT ATCGGAATGA

This corresponds to the amino acid sequence <SEQ ID 66; ORF118>: 1 ..TTGSLGGILA GGGTSLAAPY LDKAAENLGP AGKAAVNALG GAAIGYATGG 51   SGGAVVGANV DWNNRQLHPK EMALADKYAE ALKREVEKRE GRKISSQEAA 101   MRIRRQICVG WTKVPKTAIP TKASYPLSE*

Computer analysis of this amino acid sequence reveals two putative transmembrane domains.

Based on this analysis, it is predicted that this protein from N. meningitidis, and its epitopes, could be useful antigens for vaccines or diagnostics.

Example 16

The following partial DNA sequence was identified in N. meningitidis SEQ ID 67> 1 ..CAATGCCGTC TGAAAAGCTC ACAATYTTAC AGACGGCATT TGTTATGCAA 51   GTACATATAC AGATTCCCTA TATACTGCCC AGrkGCGTGC GTgGCTGAAG 101   ACACCCCCTA CGCTTGCTAT TTGrAACAGC TCCAAGTCAC CAAAGACGTC 151   AACTGGAACC AGGTACWACT GGCGTACGAC AAATGGGACT ATAAACAGGA 201   AGGCTTAACC GGAGCCGGAG CAGCGATTAT TGCGCTGGCT GTTACCGTGG 251   TTACTGCGGG CGCGGGAgCC GGAGCCGCAC TGGGcTTAAA CGGCGCGGCc 301   GCAGCGGCAA CCGATGCCGC ATTCGCCTCG CTGGCCAGCC AGGcTTCCGT 351   ATCGCTCATC AaCAACAAAG GCAATATCGG TAaCACCCTG AAAGAGCTGG 401   GCAGAAGCAG CACGGTGAAA AATCTGATGG TTGCCGTCGc tACCGCAgGC 451   GTagCcgaCA AAATCGGTGC TTCGGCACTG AACAATGTCA GCGATAAGCA 501   GTGGATCAAC AACCTGACCG TCAACCTGGC CAATGCGGGC AGTGCCGCAC 551   TGATTAATAC CGCTGTCAAC GGCGGCAGCc tgAAAGACAA TCTGGAAGCG 601   AATATCCTTG CGGCTTTGGT GAATACTGCG CATGGAGAAG CAGCCAGTAA 651   AATCAAACAG TTGGATCAGC ACTACATTAC CCACAAGATT GCCCaTGCCA 701   TAGCGGGCTG TGCGGcTGCG GCGGCGAATA AGGGCAAGTG TCAGGATGGT 751   GCGATAgGTG CGGCTGTGGG CGAGATAGTC GGGGAgGCTT TGACAAACGG 801   CAAAAATCCT GACACTTTGA CAGCTAAAgA ACGCGaACAG ATTTTGGCAT 851   ACAGCAAACT GGTTGCCGGT ACGGTAAGCG GTGTGGTCGG CGGCGATGTA 901   AATGCGGCGG CGAATGCGGC TGAGGTAGCG GTGAAAAATA ATCAGCTTAG 951   CGACAAAtGA

This corresponds to the amino acid sequence <SEQ ID 68; ORF41>: 1 ..QCRLKSSQFY RRHLLCKYIY RFPIYCPXAC VAEDTPYACY LXQLQVTKDV 51   HWNQVXLAYD KWDYKQEGLT GAGAAIIALA VTVVTAGAGA GAALGLNGAA 101   AAATDAAFAS LASQASVSLI NNKGNIGNTL KELGRSSTVK NU4VAVATAG 151   VADKIGASAL NNVSDKQWIN NLTVNLANAG SAALINTAVN GGSLKDNLEA 201   NILAALVNTA HGEAASKIKQ LDQHYITHKI AHAIAGCAAA AANKGKCQDG 251   AIGAAVGEIV GEALTNGKNP DTLTAKEREQ ILAYSKLVAG TVSGVVGGDV 301   NAAANAAEVA VKNNQLSDK*

Further work revealed the complete nucleotide sequence <SEQ ID 69>: 1 ATGCAAGTAA ATATTCAGAT TCCCTATATA CTGCCCAGAT GCGTGCGTGC 51 TGAAGACACC CCCTACGCTT GCTATTTGAA ACAGCTCCAA GTCACCAAAG 101 ACGTCAACTG GAACCAGGTA CAACTGGCGT ACGACAAATG GGACTATAAA 151 CAGGAAGGCT TAACCGGAGC CGGAGCAGCG ATTATTGCGC TGGCTGTTAC 201 CGTGGTTACT GCGGGCGCGG GAGCCGGAGC CGCACTGGGC TTAAACGGCG 251 CGGCCGCAGC GGCAACCGAT GCCGCATTCG CCTCGCTGGC CAGCCAGGCT 301 TCCGTATCGC TCATCAACAA CAAAGGCAAT ATCGGTAACA CCCTGAAAGA 351 GCTGGGCAGA AGCAGCACGG TGAAAAATCT GATGGTTGCC GTCGCTACCG 401 CAGGCGTAGC CGACAAAATC GGTGCTTCGG CACTGAACAA TGTCAGCGAT 451 AAGCAGTGGA TCAACAACCT GACCGTCAAC CTGGCCAATG CGGGCAGTGC 501 CGCACTGATT AATACCGCTG TCAACGGCGG CAGCCTGAAA GACAATCTGG 551 AAGCGAATAT CCTTGCGGCT TTGGTGAATA CTGCGCATGG AGAAGCAGCC 601 AGTAAAATCA AACAGTTGGA TCAGCACTAC ATTACCCACA AGATTGCCCA 651 TGCCATAGCG GGCTGTGCGG CTGCGGCGGC GAATAAGGGC AAGTGTCAGG 701 ATGGTGCGAT AGGTGCGGCT GTGGGCGAGA TAGTCGGGGA GGCTTTGACA 751 AACGGCAAAA ATCCTGACAC TTTGACAGCT AAAGAACGCG AACAGATTTT 801 GGCATACAGC AAACTGGTTG CCGGTACGGT AAGCGGTGTG GTCGGCGGCG 851 ATGTAAATGC GGCGGCGAAT GCGGCTGAGG TAGCGGTGAA AAATAATCAG 901 CTTAGCGACA AAGAGGGTAG AGAATTTGAT AACGAAATGA CTGCATGCGC 951 CAAACAGAAT AATCCTCAAC TGTGCAGAAA AAATACTGTA AAAAAGTATC 1001 AAAATGTTGC TGATAAAAGA CTTGCTGCTT CGATTGCAAT ATGTACGGAT 1051 ATATCCCGTA GTACTGAATG TAGAACAATC AGAAAACAAC ATTTGATCGA 1101 TAGTAGAAGC CTTCATTCAT CTTGGGAAGC AGGTCTAATT GGTAAAGATG 1151 ATGAATGGTA TAAATTATTC AGCAAATCTT ACACCCAAGC AGATTTGGCT 1201 TTACAGTCTT ATCATTTGAA TACTGCTGCT AAATCTTGGC TTCAATCGGG 1251 CAATACAAAG CCTTTATCCG AATGGATGTC CGACCAAGGT TATACACTTA 1301 TTTCAGGAGT TAATCCTAGA TTCATTCCAA TACCAAGAGG GTTTGTAAAA 1351 CAAAATACAC CTATTACTAA TGTCAAATAC CCGGAAGGCA TCAGTTTCGA 1401 TACAAACCTA AAAAGACATC TGGCAAATGC TGATGGTTTT AGTCAAAAAC 1451 AGGGCATTAA AGGAGCCCAT AACCGCACCA ATTTTATGGC AGAACTAAAT 1501 TCACGAGGAG GACGCGTAAA ATCTGAAACC CAAACIGATA TTGAAGGCAT 1551 TACCCGAATT AAATATGAGA TTCCTACACT AGACAGGACA GGTAAACCTG 1601 ATGGTGGATT TAAGGAAATT TCAAGTATAA AAACTGTTTA TAATCCTAAA 1651 AAATTTTCTG ATGATAAAAT ACTTCAAATG GCTCAAAATG CTGCTTCACA 1701 AGGATATTCA AAAGCCTCTA AAATTGCTCA AAATGAAAGA ACTAAATCAA 1751 TATCGGAAAG AAAAAATGTC ATTCAATTCT CAGAAACCTT TGACGGAATC 1801 AAATTTAGAT CATATTTTGA TGTAAATACA GGAAGAATTA CAAACATTCA 1851 CCCAGAATAA

This corresponds to the amino acid sequence <SEQ ID 70; ORF41-1>: 1 MQVNIQIPYI LPRCVRAEDT PYACYLKQLQ VTKDVNWNQV QLAYOKWDYK 51 QEGLTGAGAA IIALAVTVVT AGAGAGAALG LNGAAAAATD AAFASLASQA 101 SVSLINNKGN IGNTLKELGR SSTVKNLHVA VATAGVADKI GASALNNVSD 151 KQWINNLTVN LANAGSAALI NTAVNGGSLX DNLEANILAA LVNTAHGEAA 201 SKIKQLDQHY ITHKIAHAIA GCAAAAANKG KCQDGAIGAA VGEIVGEALT 251 NGKNPDTLTA KEREQILAYS KLVAGTVSGV VGGDVNAAAN AAEVAVKNNQ 301 LSDKLGREFD NEMTACAKQN NPQLCRKNTV KKYQNVADKR LAASIAICTD 351 ISRSTECRTI RKQHLIDSRS LHSSWEAGLX GKDDEWYKLF SKSYTQADLA 401 LQSYHLNTAA KSWLOSGNTK PLSEWNSDQG YTLISGVNPR FIPIPRGFVK 451 QHTFITNVKY PEGISFDTNL KRMLANADGF SQKQGIKGAH NRTNFNAELN 501 SRGGRVKSET QTDIEGITRI KYEIPTLDRT GKPDGGFKEI SSIKTVYNPK 551 KFSDDKILQH AQNAASQGYS KASKIAQNER TKSISERKNV IQFSETFDGI 601 KFRSYFDVNT GRITNIHPE*

Computer analysis of this amino acid sequence predicts a transmembrane domain, and homology with an ORF from N. meningitidis (strain A) was also found.

ORF41 shows 92.8% identity over a 279 aa overlap with an ORF (ORF41a) from strain A of N. meningitidis:  10        20        30        40        50        60        69 orf41.pep   YRRHLLCKYIYRFPIYCPXACVAEDTPYACYLXQLQVTKDVNWNQVXLAYDKWDYKQEGL                                 11 1111:1::11111 1111:11111111 orf41a                                 YLKQLQVAXNINWNQVQLAYDRWDYKQEGL                                         10        20        30  70        80        90       100       110       120       129 orf41.pep   TGAGAAIIALAVTVVTAGAGAGAALGLNGAAAAATDAAFASLASQASVSLINNKGNIGNT   | ||||||||||||||:|||:||:|||||| ||||||||||||||||||:|||||::|:| orf41a   TEAGAAIIALAVTVVTSGAGTGAVLGLNGAXAAATDAAFASLASQASVSFINNKGDVGKT           40        50        60        70        80        90 130       140       150       160       170       180       189 orf41.pep   LKELGRSSTVKNLMVAVATAGVADKIGASALNNVSDKQWINNLTVNLANAGSAALINTAV   |||||||||||||:||:|||||||||||||| |||||||||||||||||||||||||||| orf41a   LKELGRSSTVKNLVVAAATAGVADKIGASALXNVSDKQWINNLTVNLANAGSAALINTAV          100       110       120       130       140       150 190       200       210       220       230       240       249 orf41.pep   NGGSLKDNLEANILAALVNTAHGEAASKIKQLDQHYITHKIAHAIAGCAAAAANKGKCQD   ||||||| |||||||||||||||||||||||||||||:|||||||||||||||||||||| orf41a   NGGSLKDXLEANILAALVNTAHGEAASKIKQLDQHYIVHKIAHAIAGCAAAAANKGKCQD          160       170       180       190       200       210 250       260       270       280       290       300       309 orf41.pep   GAIGAAVGEIVGEALTNGKNPDTLTAKEREQILAYSKLVAGTVSGVVGGDVNAAANAAEV   |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| orf41a   GAIGAAVGEIVGEALTNGKNPDTLTAKEREQILAYSKLVAGTVSGVVGGDVNAAANAAEV          220       230       240       250       260       270 310       320 orf41.pep   AVKNNQLSDKX   ||||||||| orf41a   AVKNNQLSDXEGREFDNENTACAKQNXPQLCRXWIVKKYQNVADKRLAASIAICTDISRS          280       290       300       310       320       330

A partial ORF41a nucleotide sequence <SEQ ID 71> is: 1 TATCTGAAAC AGCTCCAAGT AGCGAAAAAC ATCAACTGGA ATCAGGTGCA 51 GCTTGCTTAC GACAGATGGG ACTACAAACA GGAGGGCTTA ACCGAAGCAG 101 GTGCGGCGAT TATCGCACTG GCCGTTACCG TGGTCACCTC AGGCGCAGGA 151 ACCGGAGCCG TATTGGGATT AAACGGTGCG NCCGCCGCCG CAACCGATGC 201 AGCATTCGCC TCTTTGGCCA GCCAGGCTTC CGTATCGTTC ATCAACAACA 251 AAGGCGATGT CGGCAAAACC CTGAAAGAGC TGGGCAGAAG CAGCACGGTG 301 AAAAATCTGG TGGTTGCCGC CGCTACCGCA GGCGTAGCCG ACAAAATCGG 351 CGCTTCGGCA CTGANCAATG TCAGCGATAA GCAGTGGATC AACAACCTGA 401 CCGTCAACCT AGCCAATGCG GGCAGTGCCG CACTGATTAA TACCGCTGTC 451 AACGGCGGCA GCCTGAAAGA CANTCTGGAA GCGAATATCC TTGCGGCTTT 501 GGTCAATACC GCGCATGGAG AAGCAGCCAG TAAAATCAAA CAGTTGGATC 551 AGCACTACAT AGTCCACAAG ATTGCCCATG CCATAGCGGG CTGTGCGGCA 601 GCGGCGGCGA ATAAGGGCAA GTGTCAGGAT GGTGCGATAG GTGCGGCTGT 651 GGGCGAGATA GTCGGGGAGG CTTTGACAAA CGGCAAAAAT CCTGACACTT 701 TGACAGCTAA AGAACGCGAA CAGATTTTGG CATACAGCAA ACTGGTTGCC 751 GGTACGGTAA GCGGTGTGGT CGGCGGCGAT GTAAATGCGG CGGCGAATGC 801 GGCTGAGGTA GCGGTGAAAA ATAATCAGCT TAGCGACNAA GAGGGTAGAG 851 AATTTGATAA CGAAATGACT GCATGCGCCA AACAGAATAN TCCTCAACTG 901 TGCAGAAAAA ATACTGTAAA AAAGTATCAA AATGTTGCTG ATAAAAGACT 951 TGCTGCTTCG ATTGCAATAT GTACGGATAT ATCCCGTAGT ACTGAATGTA 1001 GAACAATCAG AAAACAACAT TTGATCGATA GTAGAAGCCT TCATTCATCT 1051 TGGGAAGCAG GTCTAATTGG TAAAGATGAT GAATGGTATA AATTATTCAG 1101 CAAATCTTAC ACCCAAGCAG ATTTGGCTTT ACAGTCTTAT CATTTGAATA 1151 CTGCTGCTAA ATCTTGGCTT CAATCGGGCA ATACAAAGCC TTTATCCGAA 1201 TGGATGTCCG ACCAAGGTTA TACACTTATT TCAGGAGTTA ATCCTAGATT 1251 CATTCCAATA CCAAGAGGGT TTGTAAAACA AAATACACCT ATTACTAATG 1301 TCAAATACCC GGAAGGCATC AGTTTCGATA CAAACCTANA AAGACATCTG 1351 GCAAATGCTG ATGGTTTTAG TCAAGAACAG GGCATTAAAG GAGCCCATAA 1401 CCGCACCAAT NTTATGGCAG AACTAAATTC ACGAGGAGGA NGNGTAAAAT 1451 CTGAAACCCA NACTGATATT GAAGGCATTA CCCGAATTAA ATATGATATT 1501 CCTACACTAG ACAGGACAGG TAAACCTGAT GGTGGATTTA AGGAAATTTC 1551 AAGTATAAAA ACTGTTTATA ATCCTAAAAA NTTTTNNGAT GATAAAATAC 1601 TTCAAATGGC TCAANATGCT GNTTCACAAG GATATTCAAA AGCCTCTAAA 1651 ATTGCTCAAA ATGAAAGAAC TAAATCAATA TCGGAAAGAA AAAATGTCAT 1701 TCAATTCTCA GAAACCTTTG ACGGAATCAA ATTTAGANNN TATNTNGATG 1751 TAAATACAGG AAGAATTACA AACATTCACC CAGAATAA

This encodes a protein having the partial amino acid sequence <SEQ ID 72>: 1 YLKQLQVAKN INWNQVQLAY DRWDYKQEGL TEAGAAIIAL AVTVVTSGAG 51 TGAVLGLNGA XAAATDAAFA SLASQASVSF INNKGDVGKT LKELGRSSTV 101 KNLVVAAATA GVADKIGASA LXNVSDKQWI NNLTVNLANA GSAALINTAV 151 NGGSLKDXLE ANILAALVNT AHGEAASKIK QLDQHYIVRK IAHAIAGCAA 201 AAANKGKCQD GAIGAAVGEI VGEALTNGKN PDTLTAKERE QILAYSKLVA 251 GTVSGVVGGD VNAAANAAEV AVKNNQLSDX EGREFONENT ACAKQNXPQL 301 CRKNTVKKYQ NVADKRLAAS IAICTDISRS TECRTIRKQH LIDSRSLHSS 351 WEAGLIGKDD EWYKLFSKSY TQADLALQSY BLNTAAKSWL QSGNTKPLSE 401 VNSDQGYTLI SGVNPRFIFI PRGFVKQNTP ITNVKYPEGI SFDTNLXRHL 451 ANADGFSQEQ GIKGAHNRTN XMAELNSRGG XVKSETXTDI EGITRIKYEI 501 PTLDRTGKPD GGFKEISSIK TVYNPKXFXD DKILQMAQXA XSQGYSKASK 551 IAQNERTKSI SERKNVIQFS ETFDGIKFRX YXDVNTGRIT NIHPE*

ORF41a and ORF41-1 show 94.8% identity in 595 aa overlap:                                 10        20        30 orf41a.pep                         YLKQLQVAKNINWNQVQLAYDRWDYKQEGLTEAGAA                         |||||||:|::||||||||||:||||||||| |||| orf41-1 MQVNIQIPYILPRCVRAEDTPYACYLKQLQVTKDVNWNQVQLAYDKWDYKQEGLTGAGAA         10        20        30        40        50        60   40        50        60        70        80        90 orf41a.pep IIALAVTVVTSGAGTGAVLGLNGAXAAATDAAFASLASQASVSFINNKGDVGKTLKELGR ||||||||||:|||:||:|||||| ||||||||||||||||||:|||||::|:||||||| orf41-1 IIALAVTVVTAGAGAGAALGLNGAAAAATDAAFASLASQASVSLINNKGNIGNTLKELGR         70        80        90       100       110       120  100       110       120       130       140       150 orf41a.pep SSTVKNLVVAAATAGVADKIGASALXNVSDKQWINNLTVNLANAGSAALINTAVNGGSLK |||||||:||:|||||||||||||| |||||||||||||||||||||||||||||||||| orf41-1 SSTVKNLMVAVATAGVADKIGASALNNVSDKQWINNLTVNLANAGSAALINTAVNGGSLK        130       140       150       160       170       180  160       170       180       190       200       210 orf41a.pep DXLEANILAALVNTAHGEAASKIKQLDQHYIVHKIAHAIAGCAAAAANKGKCQDGAIGAA | |||||||||||||||||||||||||||||:|||||||||||||||||||||||||||| orf41-1 DNLEANILAALVNTAHGEAASKIKQLDQHYITHKIAHAIAGCAAAAANKGKCQDGAIGAA        190       200       210       220       230       240  220       230       240       250       260       270 orf41a.pep VGEIVGEALTNGKNPDTLTAKEREQILAYSKLVAGTVSGVVGGDVNAAANAAEVAVKNNQ |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| orf41-1 VGEIVGEALTNGKNPDTLTAKEREQILAYSKLVAGTVSGVVGGDVNAAANAAEVAVKNNQ        250       260       270       280       290       300  280       290       300       310       320       330 orf41a.pep LSDXEGREFDNEMTACAKQNXPQLCRKNTVKKYQNVADKRLAASIAICTDISRSTECRTI ||| |||||||||||||||| ||||||||||||||||||||||||||||||||||||||| orf41-1 LSDKEGREFDNEMTACAKQNNPQLCRKNTVKKYQNVADKRLAASIAICTDISRSTECRTI        310       320       330       340       350       360  340       350       360       370       380       390 orf41a.pep RKQHLIDSRSLHSSWEAGLIGKODEWYKLFSKSYTQADLALQSYHLNTAMCSWLQSGNTK |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| orf41-1 RKQHLIDSRSLHSSWEAGLIGKDDEWYKLFSKSYTQADLALQSYHLNTAAXSWLOSGNTK        370       380       390       400       410       420  400       410       420       430       440       450 orf41a.pep PLSEWMSDQGYTLISGVNPRFIPIPRGFVKQNTPITNVKYPEGISFDTNLXRHLANADGF |||||||||||||||||||||||||||||||||||||||||||||||||| ||||||||| orf41-1 PLSEWMSDQGYTLISGVNPRFIPIPRGFVKQNTPITNVKYPEGISFDTNLKRHLANADGF        430       440       450       460       470       480  460       470       480       490       500       510 orf41a.pep SQEQGIKGAHNRTNXMAELNSRGGXVKSETXTDIEGITRIKYEIPTLDRTGKPDGGFKEI ||:||||||||||| ||||||||| ||||| ||||||||||||||||||||||||||||| orf41-1 SQKQGIKGAHNRTNFMAELNSRGGRVKSETQTDIEGITRIKYEIPTLDRTGKPDGGFKEI        490       500       510       520       530       540  520       530       540       550       560       570 orf41a.pep SSIKTVYNPKXFXDDKILQMAQXAXSQGYSKASKIAQNERTKSISERKNVIQFSETFDGI |||||||||| | ||||||||| | ||||||||||||||||||||||||||||||||||| orf41-1 SSIKTVYNPKKFSDDKILQMAQNAASQGYSKASKIAQNERTKSISERKNVIQFSETFDGI        550       560       570       580       590       600  580       590 orf41a.pep KFRXYXDVNTGRITNIHPEX ||| | |||||||||||||| orf41-1 KFRSYFDVNTGRITNIHPEX        610       620

Amino acids 25-619 of ORF41-1 were amplified as described above. FIG. 6 shows plots of hydrophilicity, antigenic index, and AMPHI regions for ORF41-1.

Based on this analysis, it is predicted that this protein from N. meningitidis, and its epitopes, could be useful antigens for vaccines or diagnostics.

Example 17

The following DNA sequence was identified in N. meningitidis <SEQ ID 73> 1 ATGGCAATCA TTACATTGTA TTATTCTGTC AATGGTATTT TAAATGTATG 51 TGCAAAAGCA AAAAATATTC AAGTAGTTGC CAATAATAAG AATATGGTTC 101 TTTTTGGGTT TTTGGSmrGC ATCATCGGCG GTTCAACCAA TGCCATGTCT 151 CCCATATTGT TAATATTTTT GCTTAGCGAA ACAGAAAATA AAAATcgTAT 201 CGTAAAATCA AGCAATCTAT GCTATCTTTT GGCGAAAATT GTTCAAATAT 251 ATATGCTAAG AGACCAGTAT TGGTTATTAA ATAAGAGTGA ATACGdTTTA 301 ATATTTTTAC TGTCCGTATT GTCTGTTATT GGATTGTATG TTGGAATTCG 351 GTTAAGGACT AAGATTAGCC CAaATTTTTT TAAAATGTTA ATTTTTATTG 401 tTTTATTGGT ATTGGCtCTG AAAATCGGGC AttCGGGTTT AAtCAAACTT 451 TAA

This corresponds to the amino acid sequence <SEQ ID 74; ORF51>: 1 HAIITLYYSV NGILNVCAKA KNIQVVANNK NMVLFGFLXX IICGSTNANS 51 PILLIFLLSE TENKNRIVKS SNLCYLLAKI VQIYMLRDQY WLLNKSEYXL 101 IFLLSVLSVI GLYVGIRLRT KISPNFFKML IFIVLLVLAL KIGHSGLIKL 151 *

Further work revealed the complete nucleotide sequence <SEQ ID 75>: 1 ATGCAAGAAA TAATGCAATC TATCGTTTTT GTTGCTGCCG CAATACTGCA 51 CGGAATTACA GGCATGGGAT TTCCGATGCT CGGTACAACC GCATTGGCTT 101 TTATCATGCC ATTGTCTAAG GTTGTTGCCT TGGTGGCATT ACCAAGCCTG 151 TTAATGAGCT TGTTGGTTCT ATGCAGCAAT AACAAAAAGG GTTTTTGGCA 201 AGAGATTGTT TATTATTTAA AAACCTATAA ATTGCTTGCT ATCGGCAGCG 251 TCGTTGGCAG CATTTTGGGG GTGAAGTTGC TTTTGATACT TCCAGTGTCT 301 TGGCTGCTTT TACTGATGGC AATCATTACA TTGTATTATT CTGTCAATGG 351 TATTTTAAAT GTATGTGCAA AAGCAAAAAA TATTCAAGTA GTTGCCAATA 401 ATAAGAATAT GGTTCTTTTT GGGTTTTTGG CAGGCATCAT CGGCGGTTCA 451 ACCAATGCCA TGTCTCCCAT ATTGTTAATA TTTTTGCTTA GCGAAACAGA 501 AAATAAAAAT CGTATCGTAA AATCAAGCAA TCTATGCTAT CTTTTGGCGA 551 AAATTGTTCA AATATATATG CTAAGAGACC AGTATTGGTT ATTAAATAAG 601 AGTGAATACG GTTTAATATT TTTACTGTCC GTATTGTCTG TTATTGGATT 651 GTATGTTGGA ATTCGGTTAA GGACTAAGAT TAGCCCAAAT TTTTTTAAAA 701 TGTTAATTTT TATTGTTTTA TTGGTATTGG CTCTGAAAAT CGGGCATTCG 751 GGTTTAATCA AACTTTAA

This corresponds to the amino acid sequence <SEQ ID 76; ORF51-1>: 1 MQEIMQSIVF VAAAILHGIT GMGFPMLGTT ALAFIMPLSK VVALVALPSL 51 LMSLLVLCSN NKKGFWQEIV YYLKTYKLLA IGSVVGSILG VKLLLILPVS 101 WLLLLMAIIT LYYSVNGILN VCAKAKNIQV VANNKNNVLF GFLAGIIGGS 151 TNAMSPILLI FLLSETENKN RIVKSSNLCY LLAKIVQIYN LRDQYWLLNK 201 SEYGLIFLLS VLSVIGLYVG IRLRTKISPN FFKMLIFIVL LVLALKIGHS 251 GLIKL*

Computer analysis of this amino acid sequence reveals three putative transmembrane domains. A corresponding ORF from strain A of N. meningitidis was also identified:

Homology with a Predicted ORF from N. meningitidis (Strain A)

ORF51 shows 96.7% identity over a 150 aa overlap with an ORF (ORF51a) from strain A of N. meningitidis:                                       10        20        30 orf51.pep                               MAIITLYYSVNGILNVCAKAKNIQVVANNK                               |||||||||||||||||||||||||||||| orf51a YKLLAIGSVVGSILGVKLLLILPVSWLLLLMAIITLYYSVNGILNVCAKAKNIQVVANNK    80        90       100       110       120       130         40        50        60        70        80        90 orf51.pep NMVLFGFLXXIIGGSTNAMSPILLIFLLSETENKNRIVKSSNLCYLLAKIVQIYMLRDQY |||||||| ||||||||||||||||||||||||||||:|||||||||||||||||||||| orf51a NMVLFGFLAGIIGGSTNAMSPILLIFLLSETENKNRIAKSSNLCYLLAKIVQIYMLRDQY   140       150       160       170       180       190        100       110       120       130       140       150 orf51.pep WLLNKSEYXLIFLLSVLSVIGLYVGIRLRTKISPNFFKMLIFIVLLVLALKIGHSGLIKL |||||||| ||||||||||||||||||||||||||||||||||||||||||||:|||||| orf51a WLLNKSEYGLIFLLSVLSVIGLYVGIRLRTKISPNFFKMLIFIVLLVLALKIGYSGLIKL   200       210       220       230       240       250

ORF51-1 and ORF51a show 99.2% identity in 255 aa overlap: orf51a.pep MQEIMQSIVFVAAAILHGITGMGFPMLGTTALAFIMPLSKVVALVALPSLLMSLLVLCSN |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| orf51-1 MQEIMQSIVFVAAAILHGITGMGFPMLGTTALAFIMPLSKVVALVALPSLLMSLLVLCSN orf51a.pep NKKGFWQEIVYYLKTYKLLAIGSVVGSILGVKLLLILPVSWLLLLMAIITLYYSVNGILN |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| orf51-1 NKKGFWQEIVYYLKTYKLLAIGSVVGSILGVKLLLILPVSWLLLLMAIITLYYSVNGILN orf51a.pep VCAKAKNIQVVANNKNMVLFGFLAGIIGGSTNAMSPILLIFLLSETENKNRIAKSSNLCY ||||||||||||||||||||||||||||||||||||||||||||||||||||:||||||| orf51-1 VCAKAKNIQVVANNKNMVLFGFLAGIIGGSTNAMSPILLIFLLSETENKNRIVKSSNLCY orf51a.pep LLAKIVQIYMLRDQYWLLNKSEYGLIFLLSVLSVIGLYVGIRLRTKISPNFFKMLIFIVL |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| orf51-1 LLAKIVQIYMLRDQYWLLNKSEYGLIFLLSVLSVIGLYVGIRLRTKISPNFFKMLIFIVL orf51a.pep LVLALKIGYSGLIKLX ||||||||:||||||| orf51-1 LVLALKIGHSGLIKLX

The complete length ORF51a nucleotide sequence SEQ D 77> is: 1 ATGCAAGAAA TAATGCAATC TATCGTTTTT GTTGCTGCCG CAATACTGCA 51 CGGAATTACA GGCATGGGAT TTCCGATGCT CGGTACAACC GCATTGGCTT 101 TTATCATGCC ATTGTCTAAG GTTGTTGCCT TGGTGGCATT ACCAAGCCTG 151 TTAATGAGCT TGTTGGTTCT ATGCAGCAAT AACAAAAAGG GTTTTTGGCA 201 AGAGATTGTT TATTATTTAA AAACCTATAA ATTGCTTGCT ATCGGCAGCG 251 TCGTTGGCAG CATTTTGGGG GTGAAGTTGC TTTTGATACT TCCAGTGTCT 301 TGGCTGCTTT TACTGATGGC AATCATTACA TTGTATTATT CTGTCAATGG 351 TATTTTAAAT GTATGTGCAA AAGCAAAAAA TATTCAAGTA GTTGCCAATA 401 ATAAGAATAT GGTTCTTTTT GGGTTTTTGG CAGGCATCAT CGGCGGTTCA 451 ACCAATGCCA TGTCTCCCAT ATTGTTAATA TTTTTGCTTA GCGAAACAGA 501 GAATAAAAAT CGTATCGCAA AATCAAGCAA TCTATGCTAT CTTTTGGCAA 551 AAATTGTTCA AATATATATG CTAAGAGACC AGTATTGGTT ATThAATAAG 601 AGTGAATACG GTTTAATATT TTTACTGTCC GTATTGTCTG TTATTGGATT 651 GTATGTTGGA ATTCGGTTAA GGACTAAGAT TAGCCCAAAT TTTTTTAAAA 701 TGTTAATTTT TATTGTTTTA TTGGTATTGG CTCTGAAAAT CGGGTATTCA 751 GGTTTAATCA AACTTTAA

This encodes a protein having amino acid sequence <SEQ ID 78>: 1 MQEIMQSIVF VAAAILHGIT GMGFPNLGTT ALAFIMPLSK VVALVALPSL 51 LMSLLVLCSN NKKGFWQEIV YYLKTYKLLA IGSVVGSILG VKLLLILPVS 101 WLLLLMAIIT LYYSVNGILN VCAKAKNIQV VANNKNMVLF GFLAGIIGGS 151 TNAMSFILLI FLLSETENKN RIAXSSNLCY LLAKIVQIYM LRDQYWLLNK 201 SEYGLIFLLS VLSVIGLYVG IRLRTKISPN FFKMLIFIVL LVLALKIGYS 251 GLIKL*

Based on this analysis, it is predicted that this protein from N. meningitidis, and its epitopes, could be useful antigens for vaccines or diagnostics.

Example 18

The following partial DNA sequence was identified in N. meningitidis <SEQ ID 79> 1 ATGAGACATA TGAAAATACA AAATTATTTA CTAGTATTTA TAGTTTTACA 51 TATAGCCTTG ATAGTAATTA ATATAGTGTT TGGTTATTTT GTTTTTCTAT 101 TTGATTTTTT TGCGTTTTTG TTTTTTGCAA ACGTCTTTCT TGCTGTAAAT 151 TTATTATTTT TAGAAAAAAA CATAAAAAAC AAATTATTGT TTTTATTGCC 201 GATTTCTATT ATTATATGGA TGGTAATTCA TATTAGTATG ATAAATATAA 251 AATTTTATAA ATTTGAGCAT CAAATAAAGG AACAAAATAT ATCCTCGATT 301 ACTGGGGTGA TAAAACCACA TGATAGTTAT AATTATGTTT ATGACTCAAA 351 TGGATATGCT AAATTAAAAG ATAATCATAG ATATGGTAGG GTAATTAGAG 401 AAACACCTTA TATTGATGTA GTTGCATCTG ATGTTAAAAA TAAATCCATA 451 AGATTAAGCT TGGTTTGTGG TATTCATTCA TATGCTCCAT GTGCCAATTT 501 TATAAAATTT GTCAGG..

This corresponds to the amino acid sequence <SEQ ID 80; ORF82>: 1 MRHMKIQNYL LVFIVLHIAL IVINIVFGYF VFLFDFFAFL FFANVFLAVN 51 LLFLEKNIKN KLLFLLPISI IIWMVIHISM INIKFYKFEH QIKEQNISSI 101 TGVIKPNDSY NYVYDSNGYA KLKDWHRYGR VIRETPYIDV VASDVKNKSI 151 RLSLVCGIHS YAPCANFIKF VR..

Further work revealed the complete nucleotide sequence SEQ ID 81>: 1 ATGAGACATA TGAAAAATAA AAATTATTTA CTAGTATTTA TAGTTTTACA 51 TATAGCCTTG ATAGTAATTA ATATAGTGTT TGGTTATTTT GTTTTTCTAT 101 TTGATTTTTT TGCGTTTTTG TTTTTTGCAA ACGTCTTTCT TGCTGTAAAT 151 TTATTATTTT TAAAAAAAAA CATAAAAAAC AAATTATTGT TTTTATTGCC 201 GATTTCTATT ATTATATGGA TGGTAATTCA TATTAGTATG ATAAATATAA 251 AATTTTATAA ATTTGAGCAT CAAATAAAGG AACAAAATAT ATCCTCGATT 301 ACTGGGGTGA TAAAACCACA TGATAGTTAT AATTATGTTT ATGACTCAAA 351 TGGATATGCT AAATTAAAAG ATAATCATAG ATATGGTAAG GTAATTAGAG 401 AAACACCTTA TATTGATGTA GTTGCATCTG ATGTTAAAAA TAAATCCATA 451 AGATTAAGCT TGGTTTGTGG TATFCATTCA TATGCFCCAT GTGCCAATTT 501 TATAAAATTT GCAAAAAAAC CTGTTAAAAT TTATTTTTAT AATCAACCTC 551 AAGGAGATTT TATAGATAAT GTAATATTTG AAATTAATGA TGGAAACAAA 601 AGTTTGTACT TGTTAGATAA GTATAAAACA TTTTTTCTTA TTGAAAACAG 651 TGTTTGTATC GTATTAATTA TTTTATATTT AAAATTTAAT TTGCTTTTAT 701 ATAGGACTTA CTTCAATGAG TTGGAATAG

This corresponds to the amino acid sequence <SEQ ID 82; ORF82-1>: 1 MRHMKNKNYL LVFIVLHIAL IVINIVFGYF VFLFDFFAFL FFANVFLAVN 51 LLFLEKNIKH KLLFLLPISI IIWMVIHISH INIKFYKFEH QIKEQNISSI 101 TGVIKPHDSY NYVYDSNGYA KLKDNHRYGR VIRETPYIDV VASDVKNKSI 151 RLSLVCGIHS YAPCANFIKF AKKPVKIYFY NQPQGDFIDN VIFEINDGNK 201 SLYLLDKYKT FFLIENSVCI VLIILYLKFN LLLYRTYFNE LE*

Computer analysis of this amino acid sequence reveals a predicted leader peptide.

A corresponding ORF from strain A of N. meningitidis was also identified:

Homology with a Predicted ORF from N. meningitidis (Strain A)

ORF82 shows 97.1% identity over a 172 aa overlap with an ORF (ORF82a) from strain A of N. meningitidis:         10        20        30        40        50        60 orf82 pep MRHMKIQNYLLVFIVLHIALIVINIVFGYFVFLFDFFAFLFFANVFLAVNLLFLEKNIKN ||||| :|||||||||||:||||||||||||||||||||||||||||||||||||||||| orf82a MRHMKIKNYLLVFIVLHITLIVINIVFGYFVFLFDFFAFLFFANVFLAVNLLFLEKNIKN         10        20        30        40        50        60         70        80        90       100       110       120 orf82 pep KLLFLLPISIIIWMVIHISMINIKFYKFEHQIKEQNISSITGVIKPHDSYNYVYDSNGYA |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| orf82a KLLFLLPISIIIWMVIHISMINIKFYKFEHQIKEQNISSITGVIKFHDSYNYVYDSNGYA         70        80        90       100       110       120        130       140       150       160       170 orf82 pep KLKDNHRYGRVIRETPYIDVVASDVKNKSIRLSLVCGIHSYAPCANFIKFVR ||||||||||||||||||||||||||||||||||||||||||||||||||:: orf82a KLKDNHRYGRVIRETPYIDVVASDVKNKSIRLSLVCGIHSYAPCANFIKFAKKPVKIYFY        130       140       150       160       170 orf82a.pep MRHMKNKNYLLVFIVLHITLIVINIVFGYFVFLFDFFAFLFFANVFLAVNLLFLEKNIKN ||||||||||||||||||:||||||||||||||||||||||||||||||||||||||||| orf82-1 MRHMKNKNYLLVFIVLHIALIVINIVFGYFVFLFDFFAFLFFANVFLAVNLLFLEKNIKN orf82a.pep KLLFLLPISIIIWMVIHISMINIKFYKFEHQIKEQNISSITGVIKPHDSYNYVYDSNGYA |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| orf82-1 KLLFLLPISIIIWMVIHISMINIKFYKFEHQIKEQNISSITGVIKPHDSYNYVYDSNGYA orf82a.pep KLKDNHRYGRVIRETPYIDVVASDVKNKSIRLSLVCGIHSYAPCANFIKFAKKPVKIYFY |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| orf82-1 KLKDNHRYGRVIRETPYIDVVASDVKNKSIRLSLVCGIHSYAPCANFIKFAKKPVKIYFY orf82a.pep NQPQGDFIDNVIFEINKGKKSLYLLDKYKTFFLIENSVCIVLIILYLKFNLLLYRTYFNE |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| orf82-1 NQPQGDFIDNVIFEINKGKKSLYLLDKYKTFFLIENSVCIVLIILYLKFNLLLYRTYFNE orf82a.pep LEX ||| orf82-1 LEX

ORF82a and ORF82-1 show 99.2% identity in 242 aa overlap: orf82a.pep MRHMKNKNYLLVFIVLHITLIVINIVFGYGVFLFDFFAFLFFANVFLAVNLLFLEKNIKN ||||||||||||||||||:||||||||||||||||||||||||||||||||||||||||| orf82-1 MRHMKNKNYLLVFIVLHIALIVINIVFGYGVFLFDFFAFLFFANVFLAVNLLFLEKNIKN orf82a.pep KLLFLLPISIIIWMVIHISMINIKFYKFEHQIKEQNISSITGVIKPHDSYNYVYDSNGYA |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| orf82-1 KLLFLLPISIIIWMVIHISMINIKFYKFEHQIKEQNISSITGVIKPHDSYNYVYDSNGYA orf82a.pep KLKDNHRYGRVIRETPYIDVVASDVKNKSIRLSLVCGIHSYAPCANFIKFAKKPVKIYFY |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| orf82-1 KLKDNHRYGRVIRETPYIDVVASDVKNKSIRLSLVCGIHSYAPCANFIKFAKKPVKIYFY orf82a.pep NQPQGDFIDNVIFEINDGKKSLYLLDKYKTFFLIENSVCIVLIILYLKFNLLLYRTYFNE ||||||||||||||||||:||||||||||||||||||||||||||||||||||||||||| orf82-1 NQPQGDFIDNVIFEINDGNKSLYLLDKYKTFFLIENSVCIVLIILYLKFNLLLYRTYFNE orf82a.pep LEX ||| orf82-1 LEX

The complete length ORF82a nucleotide sequence <SEQ D 83> is: 1 ATGAGACATA TGAAAAATAA AAATTATTTA CTAGTATTTA TAGTTTTACA 51 TATAACCTTG ATAGTAATTA ATATAGTGTT TGGTTATTTT GTTTTTCTAT 101 TTGATTTTTT TGCGTTTTTG TTTTTTGCAA ACGTCTTTCT TGCTGTAAAT 151 TTATTATTTT TAGAAAAAAA CATAAAAAAC AAATTATTGT TTTTATTGCC 201 GATTTCTATT ATTATATGGA TGGTAATTCA TATTAGTATG ATAAATATAA 251 AATTTTATAA ATTTGAGCAT CAAATAAAGG AACAAAATAT ATCCTCGATT 301 ACTGGGGTGA TAAAACCACA TGATAGTTAT AATTATGTTT ATGACTCAAA 351 TGGATATGCT AAATTAAAAG ATAATCATAG ATATGGTAGG GTAATTACAG 401 AAACACCTTA TATTGATGTA GTIGCATCTG ATGTTAAAAA TAAATCCATA 451 AGATIAAGCT TGGTTTGTGG TATTCATTCA TATGCTCCAT GTGCCAATTT 501 TATAAAATTT GCAAAAAAAC CTGTTAAAAT TTATTTTTAT AATCAACCTC 551 AAGGAGATTT TATAGATAAT GTAATATTTG AAATTAATGA TGGAAAAAAA 601 AGTTTGTACT TGTTAGATAA GTATAAAACA TTTTTTCTTA TTGAAAACAG 651 TGTTTGTATC GTATTAATTA TTTTATATTT AAAATTTAAT TTGCTTTTAT 701 ATAGGACTTA CTTCAATGAG TTGGAATAG

This encodes a protein having amino acid sequence <SEQ ID 84>: 1 MRHMKNKNYL LVFIVLHITL IVINIVFGYF VFLFDFFAFL FFANVFLAVN 51 LLFLEKNIKN KLLFLLPISI IIWMVIHISM INIKFYKFEH QIKEQNISSI 101 TGVIKPHDSY NYVYDSNGYA KLKDNHRYGR VIRETPYIDV VASDVIQKSI 151 RLSLVCGIHS YAPCANFIKF AXKPVKIYFY NQPQGDFXDN VIFEINDGKK 201 SLYLLDKYKT FFLIENSVCI VLIILYLKFN LLLYRTYFNE LE*

Based on this analysis, it is predicted that this protein from N. meningitidis, and its epitopes, could be useful antigens for vaccines or diagnostics.

Example 19

The following partial DNA sequence was identified in N. meningitidis <SEQ ID 85> 1 ..ACCCCCAACA GCGTGACCGT CTTGCCGTCT TTCGGCGGAT TCGGGCGTAC 51   CGGCGCGACC ATCAATGCAG CAGGCGGGGT CGGCATGACT GCCTTTTCGA 101   CAACCTTAAT TTCCGTAGCC GAGGGCGCGG TTGTAGAGCT GCAGGCCGTG 151   AGAGCCAAAG CCGTCAATGC AACCGCCGCT TGCATTTTTA CGGTCTTGAG 201   TAAGGACATT TTCGATTTCC TTTTTATTTT CCGTTTTCAG ACGGCTGACT 251   TCCGCCTGTA TTTTCGCCAA AGCCATGCCG ACAGCGTGCG CCTTGACTTC 301   ATATTTAAAA GCTTCCGCGC GTGCCAGTTC CAGTTCGCGC GCATAGTTTT 351   GAGCCGACAA CAGCAGGGCT TGCGCCTTGT CGCGCTCCAT CTTGTCGATG 401   ACCGCCTGCA GCTTCGCAAA TGCCGACTFG TAGCCTTGAT GGTGCGACAC 451   AGCCAAGCCC GTGCCGACAA GCGCGATAAT GGCAATCGGT TGCCAGTAAT 501   TCGCCAGCAG TTTCACGAGA TTCATTCTCG ACCTCCTGAC GCTTCACGCT 551   GA

This corresponds to the amino acid sequence <SEQ ID 86; ORF124>: 1 ..TPNSVTVLPS FGGFGRTGAT INAAGGVGMT AFSTTLISVA EGAVVELQAV 51   RAKAVNATAA CIFTVLSKDI FDFLFIFRFQ TADFRLYFRQ SHADSVRLDF 101   IFKSFRACQF QFARIVLSRQ QQGLRLVALH LVDORLQLRX CRLVALMVRH 151   SQARADKRDN GNRLPVIRQQ FHEIHSRPPD ASR*

Computer analysis of this amino acid sequence predicts a transmembrane domain.

Further work revealed the complete nucleotide sequence SEQ ID 87>: 1 ATGACTGCCT TTTCGACAAC CTTAATTTCC GTAGCCGAGG GCGCGGTTGT 51 AGAGCTGCAG GCCGTGAGAG CCAAAGCCGT CAATGCAACC GCCGCTTGCA 101 TTTTTACGGT CTTGAGTAAG GACATTTTCG ATTTCCTTTT TATTTTCCGT 151 TTTCAGACGG CTGACTTCCG CCTGTTTTTT CGCCAAAGCC ATGCCGACAG 201 CGTGCGCCTT GACTTCATAT TTTTTAGCTT CCGcGCGTGC CAGTTCCAGT 251 TCGCGCGCAT AGTTTTGAGC CGACAACAGC AGGGCTTGCG CCTTGTCGCG 301 CTCCATCTTG TCGATGACCG CCTGCTGCTT CGCAAATGCC GACTTGTAGC 351 CTTGATGGTG CGACACAGCC AAGCCCGTGC CGACAAGCGC GATAATGGCA 401 ATCGGTTGCC AGTTATTCGC CAGCAGTTTC ACGAGATTCA TTCTCGACCT 451 CCTGACGCTT CACGCTGA

This corresponds to the amino acid sequence SEQ ID 88; ORF124-1>: 1 MTAFSTTLIS VAEGAVVELQ AVRAKAVNAT AACIFTVLSK DIFDFLFIFR 51 FQTADFRLFF RQSHADSVRL DFIFFSFRAC QFQFARIVLS RQQQGLRLVA 101 LHLVDDRLLL RKCRLVAIMV RHSQARADKR DNGNRLPVIR QQFHEIHSRP 151 PDASR*

A corresponding ORF from strain A of N. meningitidis was also identified:

Homology with a Predicted ORF from N. meningitidis (Strain A)

ORF124 shows 87.5% identity over a 152 aa overlap with an ORF (ORF124a) from strain A of N. meningitidis:         10        20        30        40        50        60 orf124.pep TPNSVTVLPSFGGFGRTGATINAAGGVGMTAFSTTLISVAEGAVVELQAVRAKAVNATAA                             |||||||||||||||:|||||| |||||:||| orf124a                             MTAFSTTLISVAEGALVELQAVMAKAVNTTAA                                     10        20        30         70        80        90       100       110       120 orf124.pep CIFTVLSKDIFDFLFIFRFQTADFRLYFRQSHADSVRLDFIFKSFRACQFQFARIVLSRQ ||||||||||||||||||||||||||:|||||||:||||||| |||:  |||| :||||| orf124a CIFTVLSKDIFDFLFIFRFQTADFRLFFRQSHADGVRLDFIFFSFRTRLFQFAGVVLSRQ       40        50        60        70        80        90        130       140       150       160       170       180 orf124.pep QQGLRLVALHLVDDRLQLRKCRLVALMVRHSQARADKRDNGNRLPVIRQQFHEIHSRPPD ||||||||||:::||| ||| ||||||||| |:||||||:|||||||||||||||||||| orf124a QQGLRLVALHFLNDRLLLRKSRLVALMVRHRQTRADKRDDGNRLPVIRQQFHEIHSRPPD      100       110       120       130       140       150 orf124.pep ASRX : orf124a VX

ORF124a and ORF124-1 show 89.5% identity in 152 aa overlap: orf124-1.pep MTAFSTTLISVAEGAVVELQAVRAKAVNATAACIFTVLSKDIFDFLFIFRFQTADFRLFF |||||||||||||||:|||||| |||||:||||||||||||||||||||||||||||||| orf124a MTAFSTTLISVAEGALVELQAVMAKAVNTTAACIFTVLSKDIFDFLFIFRFQTADFRLFF orf124-1.pep RQSHADSVRLDFIFFSFRACQFQFARIVLSRQQQGLRLVALHLVDDRLLLRKCRLVALMV ||||||:|||||||||||:  |||| :|||||||||||||||:::||||||| ||||||| orf124a RQSHADGVRLDFIFFSFRTRLFQFAGVVLSRQQQGLRLVALHFLNDRLLLRKSRLVALMV orf124-1.pep RHSQARADKRDNGNRLPVIRQQFHEIHSRPPDASRX || |:||||||:||||||||||||||||||||: orf124a RHRQTRADKRDDGNRLPVIRQQFHEIHSRPPDVX

The complete length ORF124a nucleotide sequence <SEQ ID 89> is: 1 ATGACCGCCT TTTCGACAAC CTTAATTTCC GTAGCCGAGG GCGCGCTTGT 51 AGAGCTGCAA GCCGTGATGG CCAAAGCCGT CAATACAACC GCCGCCTGCA 101 TTTTTACGGT CTTGAGTAAG GACATTTTCG ATTTCCTTTT TATTTTCCGT 151 TTTCAGACGG CTGACTTCCG CCTGTTTTTT CGCCAAAGCC ATGCCGACGG 201 CGTGCGCCTT GACTTCATAT TTTTTAGCTT CCGCACGCGC CTGTTCCAGT 251 TCGCGGGCGT AGTTTTGAGC CGACAACAGC AGGGCTTGCG CCTTGTCGCG 301 CTTCATTTTC TCAATGACCG CCTGCTGCTT CGCAAAAGCC GACTTGTAGC 351 CTTGATGGTG CGACACCGCC AAACCCGTGC CGACAAGCGC GATGATGGCA 401 ATCGGTTGCC AGTTATTCGC CAGCAGTTTC ACGAGATTCA TTCTCGACCT 451 CCTGACGTTT GA

This encodes a protein having amino acid sequence <SEQ ID 90>: 1 MTAFSTTLIS VAEGALVELQ AVNAXAVNTT AACIFTVLSK DIFDFLFIFR 51 FQTADFRLFF RQSHADGVRL DFIFFSFRTR LFQFAGVVLS RQQQGLRLVA 101 LHFLNDELLL RKSRLVALHV RHRQTRADKR DDGNRLPVIR QQFHEIHSRP 151 PDV*

ORF124-1 was amplified as described above. FIG. 7 shows plots of hydrophilicity, antigenic index, and AMPHI regions for ORF124-1.

Based on this analysis, it is predicted that this protein from N. meningitidis, and its epitopes, could be useful antigens for vaccines or diagnostics.

Example 20

Table III lists several Neisseria strains which were used to assess the conservation of the sequence of ORF 40 among different strains. TABLE III List of Neisseria Strains Used for Gene Variability Study of ORF 40 Identification number Strains Source/reference Group B zn02_1 BZ198 R. Moxon/Seiler et al., 1996 zn03_1 NG3/88 R. Moxon/Seiler et al., 1996 zn04_1 297-0 R. Moxon/Seiler et al., 1996 zn06_1 BZ147 R. Moxon/Seiler et al., 1996 zn07_1 BZ169 R. Moxon/Seiler et al., 1996 zn08_1 528 R. Moxon/Seiler et al., 1996 zn10_1 BZ133 R. Moxon/Seiler et al., 1996 zn11_1ass NGE31 R. Moxon/Seiler et al., 1996 zn14_1 NGH38 R. Moxon/Seiler et al., 1996 zn16_1 NGH15 R. Moxon/Seiler et al., 1996 zn18_1 BZ232 R. Moxon/Seiler et al., 1996 zn19_1 BZ83 R. Moxon/Seiler et al., 1996 zn20_1 44/76 R. Moxon/Seiler et al., 1996 zn21_1 MC58 R. Moxon Group A zn22_1 205900 R. Moxon zn23_1 F6124 R. Moxon z2491_1 Z2491 R. Moxon/Maiden et al., 1998 Group C zn24_1 90/18311 R. Moxon zn25_1ass 93/4286 R. Moxon Others zn28_1ass 860800 (group Y) R. Moxon/Maiden et al., 1998 zn29_1ass E32 (group Z) R. Moxon/Maiden et al., 1998 References: Seiler A. et al., Mol. Microbiol., 1996, 19(4): 841-856. Maiden et al., Proc. Natl. Acad. Sci. USA, 1998, 95: 3140-3145.

The amino acid sequences for each listed strain are as follows: >Z2491 <SEQ ID 91> MNKIYRIIWNSALNAWVAVSELTRNHTKRASATVKTAVLATLLFATVQANATDEDEEEEL ESVQRSVVGSIQASMEGSGELETISLSMTNDSKEFVDPYIVVTLKAGDNLKIKQNTNENT NASSFTYSLKKDLTGLINVETEKLSFGANGKKVNIISDTKGLNFAKETAGTNGDTTVHLN GIGSTLTDTLAGSSASHVDAGNQSTHYTRAASIKDVLNAGWNIKGVKTGSTTGQSENVDF VRTYDTVEFLSADTKTTTVNVESKDNGKRTEVKIGAKTSVIKEKDGKLVTGKGKGENGSS TDEGEGLVTAKEVIDAVNKAGWRMKTTTANGQTGQADKFETVTSGTNVTFASGKGTTATV SKDDQGNITVMYDVNVGDALNVNQLQNSGWNLDSKAVAGSSGKISGNVSPSKADEMDETV NINAGNNIEISRNGKNIDIATSNAPQFSSVSLGAGADAPTLSVDDEGALNVGSKDANKPV RINVAPGVKEGDVTNVAQLKGVAQNLNNRIDNVWDGNARAGIAQAIATAGLVQAYLPGKS MMAIGGGTYRGEAGYAIGYSSISDGGNWIIKGTASGNSRGHFGASASVGYQW* >ZN02_1 <SEQ ID 92> MNKIYRIIWNSALNAWVVVSELTRNHTKRASATVATAVLATLLFATVQANATDDDDLYLE PVQRTAVLSFRSDKEGTGEKEGTEDSHGGAVYFDEKRVLKAGAITLKAGDNLKIKQNTNE NTNDSSFTYSLKKDLTDLTSVETEKLSFGAAGNKVNITSDTKGLNFAKETAGTAGDPTVH LNGIGSTLTDTLLNTGATTNVTNDNVTDDEKKRAASVKDVLNAGWNIKGVKPGTTASDNV DFVRTYDTVEFLSADTKTTTNVSKDNGKKTEVICIGAIVTSVIKEKDGKLVTGKGKDENG SSTDEGEGLVTAKEVIDAVNKAGWRMKTTTANGQTGQADKFETVTSGTNVTFASGKGTTA TVSKODQGNITVRYDVNVGDALNVNQLQNSGWNLDSKAVAGSSGKVISGNVSPSKGRMDE TVNINAGNNIEITRNGKNIDIATSMAPQFSSVSLGAGADAPTLSVDDEGALNVGSKDTNK PVRITNVAPGVKEGDVTNVAQLKGVAQNLNNRIDNVDGNARAGIAQAIATAGLVQAYLPG KSMMAIGGDTYRGEAGYAIGYSSISDGGNWIIKGTASGNSRGHFGASASVGYQW* >ZN03_1 <SEQ ID 93> MNKIYRIIWNSALNAWVAVSELTRNHTKRASATVATAVLATLLFATVQASTTDDDDLYLE PVQRTAPVLSFHADSEGTGEKEVTEDSNWGVYFDKKGVLTAGTITLKAGDNLKIKQNTDE NTNDSSFTYSLKKDLTDLTSVETEKLSFGANGNKVNITSDTKGLNFAKETAGTNGDPTVH LNGIGSTLTDTLLNTGATTNVTNDNVTDDEKKRAASVKDVLNAGWNIKGVKPGTTASDNV DFVRTYDTVEFLSADTRTTTVNVESKDNGKKTEVKIGAKTSVIKEKDGKLVTGKGKDENG SSTDEGEGLVTAKEVIDAVNKAGWRMKTTTANGQTGQADKFETVTSGTNVTFASGKGTTA TVSKDDQGNITVKYDVNVGDALNVNQLQNSGWNLDSKAVAGSSGKVISGNVSPSKGKMDE TVNINAGNNIEITRNGKNIDIATSMAPQFSSVSLGAGADAPTLSVDDEGALNVGSKDTNK PVRITNVAPGVKEGDVTNVAQLKGVAQNLNNRIDNVDGNARAGIAQAIATAGLVQAYLPG KSMMAIGGDTYRGEAGYAIGYSSISDGGNMIIKGTASGNSRGHFGASASVGYQW* >ZN04_1 <SEQ ID 94> MNKIYRIIWNSALNAWVAVSELTRNHTKRASATVATAVLATLLFATVQASTTDDDDLYLE PVQRTAPVLSFHADSEGTGEKEVTEDSNWGVYFDKKGVLTAGTITLKAGDNLKIKQNTDE NTNDSSFTYSLKKDLTDLTSVETEKLSFGANGNKVNITSDTKGLNFAKETAGTNGDPTVH LNGIGSTLTDTLLNTGATTNVTNDNVTDDEKKRAASVKDVLNAGWNIKGVKPGTTASDNV DFVRTYDTVEFLSADTRTTTVNVESKDNGKKTEVKIGAKTSVIKEKDGKLVTGKGKDENG SSTDEGEGLVTAKEVIDAVNKAGWRMKTTTANGQTGQADKFETVTSGTNVTFASGKGTTA TVSKDDQGNITVKYDVNVGDALNVNQLQNSGWNLDSKAVAGSSGKVISGNVSPSKGKMDE TVNINAGNNIEITRNGKNIDIATSMAPQFSSVSLGAGADAPTLSVDDEGALNVGSKDTNK PVRITNVAPGVKEGDVTNVAQLKGVAQNLNNRIDNVDGNARAGIAQAIATAGLVQAYLPG KSMMAIGGDTYRGEAGYAIGYSSISDGGNMIIKGTASGNSRGHFGASASVGYQW* >ZN06_1 <SEQ ID 95> MNKIYRIIWNSALNAWVAVSELTRNHTKRASATVKTAVLATLLFATVQANATDEDEEEEL ESVQRSVVGSIQASMEGSGELETISLSMTNDSKEFVDPYIVVTLKAGDNLKIKQNTNENT NASSFTYSLKKDLTGLINVETEKLSFGANGKKVNIISDTKGLNFAKETAGTNGDTTVHLN GIGSTLTDTLAGSSASHVDAGNQSTHYTRAASIKDVLNAGWNIKGVKTGSTTGQSENVDF VRTYDTVEFLSADTKTTTVNVESKDNGKRTEVKIGAKTSVIKEKDGKLVTGKGKGENGSS TDEGEGLVTAKEVIDAVNKAGWRMKTTTANGQTGQADKFETVTSGTNVTFASGKGTTATV SKDDQGNITVMYDVNVGDALNVNQLQNSGWNLDSKAVAGSSGKISGNVSPSKADEMDETV NINAGNNIEISRNGKNIDIATSNAPQFSSVSLGAGADAPTLSVDDEGALNVGSKDANKPV RINVAPGVKEGDVTNVAQLKGVAQNLNNRIDNVWDGNARAGIAQAIATAGLVQAYLPGKS MMAIGGGTYRGEAGYAIGYSSISDGGNWIIKGTASGNSRGHFGASASVGYQW* >ZN07_1 <SEQ ID 96> MNKIYRIIWNSALNAWVAVSELTRNHTKRASATVKTAVLATLLFATVQANATDEDEEEEL ESVQRSVVGSIQASMEGSGELETISLSMTNDSKEFVDPYIVVTLKAGDNLKIKQNTNENT NASSFTYSLKKDLTGLINVETEKLSFGANGKKVNIISDTKGLNFAKETAGTNGDTTVHLN GIGSTLTDTLAGSSASHVDAGNQSTHYTRAASIKDVLNAGWNIKGVKTGSTTGQSENVDF VRTYDTVEFLSADTKTTTVNVESKDNGKRTEVKIGAKTSVIKEKDGKLVTGKGKGENGSS TDEGEGLVTAKEVIDAVNKAGWRMKTTTANGQTGQADKFETVTSGTNVTFASGKGTTATV SKDDQGNITVMYDVNVGDALNVNQLQNSGWNLDSKAVAGSSGKISGNVSPSKADEMDETV NINAGNNIEISRNGKNIDIATSNAPQFSSVSLGAGADAPTLSVDDEGALNVGSKDANKPV RINVAPGVKEGDVTNVAQLKGVAQNLNNRIDNVWDGNARAGIAQAIATAGLVQAYLPGKS MMAIGGGTYRGEAGYAIGYSSISDGGNWIIKGTASGNSRGHFGASASVGYQW* >ZN08_1 <SEQ ID 97> MNKIYRIIWNSALNAWVVVSELTRNHTKRASATVETAVLATLLFATVQANATDTDEDDEL EPVVRSALVLQFMIDKEGNGEIESTGDIGWSIYYDDHNTLHGATVTLKAGDNLKIKQNTD ENTNASSFTYSLKKDLTDLTSVGTEELSFGANGNKVNITSDTKGLNFAKKTAGTNGDTTV HLNGIGSTLTDTLAGSSASHVDAGNQSTHYTRAASIKDVLNAGWNIKGVKTGSTTGQSEN VDFVRTYDTVEFLSADTKTTTVNVESKDNGKRTEVKIGAKTSVIKEKDGKLVTGKGKGEN GSSTEDGEGELVTAKEVIDAVNKAGWRMKTTANGQTGQADKFETVTSGTNVTFASGKGTT ATVSKDDQGNITVKYDVNVGDALNVNQLQNSGWNLDSKAVAGSSGKISGNAVSPSKGKMD ETVNINAGNNIEITRNGKNIDIATSMTPQFSSVSLGAGADAPLTLSVDDEALNVGSKDAN KPVRITNVAPGVKEGDVTNVAQLKGVAQNLNNHIDNVDGNARAGIAQAIATAGLVQAYLP GKSMMAIGGGTYRGEAGYAIGYSSISDGGNWIIKGTASGNSRGHFGASASVGYQW* >ZN10_1 <SEQ ID 98> MNKIYRIIWNSALNAWVAVSELTRNHTKRASATVKTAVLATLLFATVQANATDEDEEEEL ESVQRSVVGSIQASMEGSGELETISLSMTNDSKEFVDPYIVVTLKAGDNLKIKQNTNENT NASSFTYSLKKDLTGLINVETEKLSFGANGKKVNIISDTKGLNFAKETAGTNGDTTVHLN GIGSTLTDTLAGSSASHVDAGNQSTHYTRAASIKDVLNAGWNIKGVKTGSTTGQSENVDF VRTYDTVEFLSADTKTTTVNVESKDNGKRTEVKIGAKTSVIKEKDGKLVTGKGKGENGSS TDEGEGLVTAKEVIDAVNKAGWRMKTTTANGQTGQADKFETVTSGTNVTFASGKGTTATV SKDDQGNITVMYDVNVGDALNVNQLQNSGWNLDSKAVAGSSGKVISGNVSPSKGKMDETV NINAGNNIEISRNGKNIDIATSMAPQFSSVSLGAGADAPTLSVDDEGALNVGSKDANKPV RITNVAPGVKEGDVTNVAQLKGVAQNLNNRIDNVDGNARAGIAQAIATAGLVQAYLPGKS MMAIGGGYTRGEAGYAIGYSSISDGGNWIIKGTASGNSRGHFGASASVGYQW* >ZN11_1 ASS <SEQ ID 99> MNKIYRIIWNSALNAWVAVSELTRNHTKRASATVATAVLATLLFATVQASTTDDDDLYLE PVQRTAPVLSFHADSEGTGEKEVTEDSNWGVYFDKKGVLTAGTITLKAGDNLKIKQNTDE NTNDSSFTYSLKKDLTDLTSVETEKLSFGANGNKVNITSDTKGLNFAKETAGTNGDPTVH LNGIGSTLTDTLLNTGATTNVTNDNVTDDEKKRAASVKDVLNAGWNIKGVKPGTTASDNV DFVRTYDTVEFLSADTRTTTVNVESKDNGKKTEVKIGAKTSVIKEKDGKLVTGKGKDENG SSTDEGEGLVTAKEVIDAVNKAGWRMKTTTANGQTGQADKFETVTSGTNVTFASGKGTTA TVSKDDQGNITVKYDVNVGDALNVNQLQNSGWNLDSKAVAGSSGKVISGNVSPSKGKMDE TVNINAGNNIEITRNGKNIDIATSMAPQFSSVSLGAGADAPTLSVDDEGALNVGSKDTNK PVRITNVAPGVKEGDVTNVAQLKGVAQNLNNRIDNVDGNARAGIAQAIATAGLVQAYLPG KSMMAIGGDTYRGEAGYAIGYSSISDGGNMIIKGTASGNSRGHFGASASVGYQW* >ZN14_1 <SEQ ID 100> MNKIYRIIWNSALNAWVVVSELTRNHTKRASATVETAVLATLLFATVQANATDTDEDDEL EPVVRSALVLQFMIDKEGNGEIESTGDIGWSIYYDDHNTLHGATVTLKAGDNLKIKQNTD ENTNASSFTYSLKKDLTDLTSVGTEELSFGANGNKVNITSDTKGLNFAKKTAGTNGDTTV HLNGIGSTLTDTLAGSSASHVDAGNQSTHYTRAASIKDVLNAGWNIKGVKTGSTTGQSEN VDFVRTYDTVEFLSADTKTTTVNVESKDNGKRTEVKIGAKTSVIKEKDGKLVTGKGKGEN GSSTEDGEGELVTAKEVIDAVNKAGWRMKTTANGQTGQADKFETVTSGTNVTFASGKGTT ATVSKDDQGNITVKYDVNVGDALNVNQLQNSGWNLDSKAVAGSSGKISGNAVSPSKGKMD ETVNINAGNNIEITRNGKNIDIATSMTPQFSSVSLGAGADAPLTLSVDDEALNVGSKDAN KPVRITNVAPGVKEGDVTNVAQLKGVAQNLNNHIDNVDGNARAGIAQAIATAGLVQAYLP GKSMMAIGGGTYRGEAGYAIGYSSISDGGNWIIKGTASGNSRGHFGASASVGYQW* >ZN16_1 <SEQ ID 101> MNKIYRIIWNSALNAWVVVSELTRNHTKRASATVATAVLATLLFATVQANATDDDDLYLE PVQRTAVVLSFRSDKEGTGEKEGTEDSNWAVYFDEKRVLKAGAITLKAGDNLKIKQNTNE NTNENTNDSSFTYSLKKDLTDLTSVETEKLSFGANGNKVNITSDTKGLNFAKETAGTNGD PTVHLNGIGSTLTDTLLNTGATTNVTNDNVTDDEKKRAASVKDVLNAGWNIKGVKPGTTA SDNVDFVRTYDTVEFLSADTKTTTVNVESKDNGKKTEVKIGAKTSVIKEKDGKLVTGKGK DENGSSTDEGEGLVTAKEVIDAVNKAGWRMKTTTANGQTGQADKFETVTSGTKVTFASGN GTTATVSKDDQGNITVKYDVNVGDALNVNQLQNSGWNLDSKAVAGSSGKVISGNVSPSKG KMDETVNINAGNNIEITRNGKNIDIATSMTPQFSSVSLGAGADAPTLSVDDEGALNVGSK DANKPVRITNVAPGVKEGDVTNVAQLKGVAQNLNNRIDNVDGNARAGIAQAIATAGLAQA YLPGKSMMAIGGGTYRGEAGYAIGYSSISDTGNWVIKGTASGNSRGHFGASASVGYQW* >ZN18_1 <SEQ ID 102> MNKIYRIIWNSALNAWVAVSELTRNHTKRASATVATAVLATLLFATVQASTTDDDDLYLE PVQRTAPVLSFHADSEGTGEKEVTEDSNWGVYFDKKGVLTAGTITLKAGDNLKIKQNTDE NTNDSSFTYSLKKDLTDLTSVETEKLSFGANGNKVNITSDTKGLNFAKETAGTNGDPTVH LNGIGSTLTDTLLNTGATTNVTNDNVTDDEKKRAASVKDVLNAGWNIKGVKPGTTASDNV DFVRTYDTVEFLSADTRTTTVNVESKDNGKKTEVKIGAKTSVIKEKDGKLVTGKGKDENG SSTDEGEGLVTAKEVIDAVNKAGWRMKTTTANGQTGQADKFETVTSGTNVTFASGKGTTA TVSKDDQGNITVKYDVNVGDALNVNQLQNSGWNLDSKAVAGSSGKVISGNVSPSKGKMDE TVNINAGNNIEITRNGKNIDIATSMAPQFSSVSLGAGADAPTLSVDDEGALNVGSKDTNK PVRITNVAPGVKEGDVTNVAQLKGVAQNLNNRIDNVDGNARAGIAQAIATAGLVQAYLPG KSMMAIGGDTYRGEAGYAIGYSSISDGGNMIIKGTASGNSRGHFGASASVGYQW* >ZN19_1 <SEQ ID 103> MNKIYRIIWNSALNAWVVVSELTRNHTKRASATVKTAVLATLLFATVQASANNEEQEEDL YLDPVQRTVAVLIVNSDKEGTGEKEKVEENSDWAVYFNEKGVLTAREITLKAGDNLKIKQ NGTNFTYSLKKDLTDLTSVGTEKLSFSANGNKVNITSDTKGLNFAKETAGTNGDTTVHLN GIGSTLTDTLLNTGATTNVTNDNVTDDEKKRAASVKDVLNAGWNIKGVKPGTTASDNVDF VRTYDTVEFLSADTKTTTVNVESKDNGKKTEVKIGAKTSVIKEKDGKLVTGKDKGENGSS TDEGEGLVTAKEVIDAVNKAGWRMKTTTANGQTGQADKFETVTSGTNVTFASGKGTTATV SKDDQGNITVMYDVNVGDALNVNQLQNSGWNLDSKAVAGSSGKVISGNVSPSKGKMDETV NINAGNNIEITRNGKNIDIATSMTPQFSSVSLGAGADAPTLSVDGDALNVGSKKDNKPVR ITNVAPGVKEGDVTNVAQLKGVAQNLNNRIDNVDGNARAGIAQAIATAGLVQAYLPGKSM MAIGGGTYRGEAGYAIGYSSISDGGNWIIKGTASGNSRGHFGASASVGYQW* >ZN20_1 <SEQ ID 104> MNKIYRIIWNSALNAWVVVSELTRNHTKRASATVKTAVLATLLFATVQASANNEEQEEDL YLDPVQRTVAVLIVNSDKEGTGEKEKVEENSDWAVYFNEKGVLTAREITLKAGDNLKIKQ NGTNFTYSLKKDLTDLTSVGTEKLSFSANGNKVNITSDTKGLNFAKETAGTNGDTTVHLN GIGSTLTDTLLNTGATTNVTNDNVTDDEKKRAASVKDVLNAGWNIKGVKPGTTASDNVDF VRTYDTVEFLSADTKTTTVNVESKDNGKKTEVKIGAKTSVIKEKDGKLVTGKDKGENGSS TDEGEGLVTAKEVIDAVNKAGWRMKTTTANGQTGQADKFETVTSGTNVTFASGKGTTATV SKDDQGNITVMYDVNVGDALNVNQLQNSGWNLDSKAVAGSSGKVISGNVSPSKGKMDETV NINAGNNIEITRNGKNIDIATSMTPQFSSVSLGAGADAPTLSVDGDALNVGSKKDNKPVR ITNVAPGVKEGDVTNVAQLKGVAQNLNNRIDNVDGNARAGIAQAIATAGLVQAYLPGKSM MAIGGGTYRGEAGYAIGYSSISDGGNWIIKGTASGNSRGHFGASASVGYQW* >ZN21_1 <SEQ ID 105> MNKIYRIIWNSALNAWVVVSELTRNHTKRASATVKTAVLATLLFATVQASANNEEQEEDL YLDPVQRTVAVLIVNSDKEGTGEKEKVEENSDWAVYFNEKGVLTAREITLKAGDNLKIKQ NGTNFTYSLKKDLTDLTSVGTEKLSFSANGNKVNITSDTKGLNFAKETAGTNGDTTVHLN GIGSTLTDTLLNTGATTNVTNDNVTDDEKKRAASVKDVLNAGWNIKGVKPGTTASDNVDF VRTYDTVEFLSADTKTTTVNVESKDNGKKTEVKIGAKTSVIKEKDGKLVTGKDKGENGSS TDEGEGLVTAKEVIDAVNKAGWRMKTTTANGQTGQADKFETVTSGTNVTFASGKGTTATV SKDDQGNITVMYDVNVGDALNVNQLQNSGWNLDSKAVAGSSGKVISGNVSPSKGKMDETV NINAGNNIEITRNGKNIDIATSMTPQFSSVSLGAGADAPTLSVDGDALNVGSKKDNKPVR ITNVAPGVKEGDVTNVAQLKGVAQNLNNRIDNVDGNARAGIAQAIATAGLVQAYLPGKSM MAIGGGTYRGEAGYAIGYSSISDGGNWIIKGTASGNSRGHFGASASVGYQW* >ZN22_1 <SEQ ID 106> MNKIYRIIWNSALNAWVAVSELTRNHTKRASATVKTAVLATLLFATVQANATDEDEEEEL ESVQRSVVGSIQASMEGSGELETISLSMTNDSKEFVDPYIVVTLKAGDNLKIKQNTNENT NASSFTYSLKKDLTGLINVETEKLSFGANGKKVNIISDTKGLNFAKETAGTNGDTTVHLN GIGSTLTDTLAGSSASHVDAGNQSTHYTRAASIKDVLNAGWNIKGVKTGSTTGQSENVDF VRTYDTVEFLSADTKTTTVNVESKDNGKRTEVKIGAKTSVIKEKDGKLVTGKGKGENGSS TDEGEGLVTAKEVIDAVNKAGWRMKTTTANGQTGQADKFETVTSGTNVTFASGKGTTATV SKDDQGNITVMYDVNVGDALNVNQLQNSGWNLDSKAVAGSSGKVISGNVSPSKGKMDETV NINAGNNIEISRNGKNIDIATSMAPQFSSVSLGAGADAPTLSVDDEGALNVGSKDANKPV RITNVAPGVKEGDVTNVAQLKGVAQNLNNRIDNVDGNARAGIAQAIATAGLVQAYLPGKS MMAIGGGYTRGEAGYAIGYSSISDGGNWIIKGTASGNSRGHFGASASVGYQW* >ZN23_1 <SEQ ID 107> MNKIYRIIWNSALNAWVAVSELTRNHTKRASATVKTAVLATLLFATVQANATDEDEEEEL ESVQRSVVGSIQASMEGSGELETISLSMTNDSKEFVDPYIVVTLKAGDNLKIKQNTNENT NASSFTYSLKKDLTGLINVETEKLSFGANGKKVNIISDTKGLNFAKETAGTNGDTTVHLN GIGSTLTDTLAGSSASHVDAGNQSTHYTRAASIKDVLNAGWNIKGVKTGSTTGQSENVDF VRTYDTVEFLSADTKTTTVNVESKDNGKRTEVKIGAKTSVIKEKDGKLVTGKGKGENGSS TDEGEGLVTAKEVIDAVNKAGWRMKTTTANGQTGQADKFETVTSGTNVTFASGKGTTATV SKDDQGNITVMYDVNVGDALNVNQLQNSGWNLDSKAVAGSSGKVISGNVSPSKGKMDETV NINAGNNIEISRNGKNIDIATSMAPQFSSVSLGAGADAPTLSVDDEGALNVGSKDANKPV RITNVAPGVKEGDVTNVAQLKGVAQNLNNRIDNVDGNARAGIAQAIATAGLVQAYLPGKS MMAIGGGYTRGEAGYAIGYSSISDGGNWIIKGTASGNSRGHFGASASVGYQW* >ZN24_1 <SEQ ID 108> MNKIYRIIWNSALNAWVVVSELTRNHTKRASATVATAVLATLLSATVQANATDTDEDEEL ESVVRSALVLQFMIDKEGNGEIESTGDIGWSIYYDDHNTLHGATVTLKAGDNLKIKQSGK DFTYSLKKELKDLTSVETEKLSFGANGNKVNITSDTKGLNFAKETAGTNGDPTVHLNGIG STLTDTLAGSSASHVDAGNQSTHYTRAASIKDVLNAGWNIKGVKTGSTTGQSENVDFVRT YDTVEFLSADTKTTTVNVESKDNGKRTEVKIGAKTSVIKEKDGKLVTGKGKGENGSSTDE GEGLVTAKEVIDAVNKAGWRMKTTTANGQTGQADKFETVTSGTKVTFASGNGTTATVSKD DQGNITVKYDVNVGDALNVNQLQNSGWNLDSKAVAGSSGKVISGNVSPSKGKMDETVNIN AGNNIEITRNGKNIDIATSMTPQFSSVSLGAGADAPTLSVDDEGALNVGSKDANKPVRIT NVAPGVKEGDVTNVAQLKGVAQNLNNRIDNVDGNARAGIAQAIATAGLAQAYLPGKSMMA IGGGTYRGEAGYAIGYSSISDTGNWVIKGTASGNSRGHFGTSASVGYQW* >ZN25_ASS <SEQ ID 109> MNKIYRIIWNSALNAWVVVSELTRNHTKRASATVATAVLATLLSATVQANATDTDEDEEL ESVVRSALVLQFMIDKEGNGEIESTGDIGWSIYYDDHNTLHGATVTLKAGDNLKIKQSGK DFTYSLKKELKDLTSVETEKLSFGANGNKVNITSDTKGLNFAKETAGTNGDPTVHLNGIG STLTDTLAGSSASHVDAGNQSTHYTRAASIKDVLNAGWNIKGVKTGSTTGQSENVDFVRT YDTVEFLSADTKTTTVNVESKDNGKRTEVKIGAKTSVIKEKDGKLVTGKGKGENGSSTDE GEGLVTAKEVIDAVNKAGWRMKTTTANGQTGQADKFETVTSGTKVTFASGNGTTATVSKD DQGNITVKYDVNVGDALNVNQLQNSGWNLDSKAVAGSSGKVISGNVSPSKGKMDETVNIN AGNNIEITRNGKNIDIATSMTPQFSSVSLGAGADAPTLSVDDEGALNVGSKDANKPVRIT NVAPGVKEGDVTNVAQLKGVAQNLNNRIDNVDGNARAGIAQAIATAGLAQAYLPGKSMMA IGGGTYRGEAGYAIGYSSISDTGNWVIKGTASGNSRGHFGTSASVGYQW* >ZN28_ASS <SEQ ID 110> MNKIYRIIWNSALNAWVAVSELTRNHTKRASATVKTAVLATLLFATVQANATDEDEEEEL ESVQRSVVGSIQASMEGSGELETISLSMTNDSKEFVDPYIVVTLKAGDNLKIKQNTNENT NASSFTYSLKKDLTGLINVETEKLSFGANGKKVNIISDTKGLNFAKETAGTNGDTTVHLN GIGSTLTDTLAGSSASHVDAGNQSTHYTRAASIKDVLNAGWNIKGVKTGSTTGQSENVDF VRTYDTVEFLSADTKTTTVNVESKDNGKRTEVKIGAKTSVIKEKDGKLVTGKGKGENGSS TDEGEGLVTAKEVIDAVNKAGWRMKTTTANGQTGQADKFETVTSGTNVTFASGKGTTATV SKDDQGNITVMYDVNVGDALNVNQLQNSGWNLDSKAVAGSSGKVISGNVSPSKGKMDETV NINAGNNIEISRNGKNIDIATSMAPQFSSVSLGAGADAPTLSVDDEGALNVGSKDANKPV RITNVAPGVKEGDVTNVAQLKGVAQNLNNRIDNVDGNARAGIAQAIATAGLVQAYLPGKS MMAIGGGYTRGEAGYAIGYSSISDGGNWIIKGTASGNSRGHFGASASVGYQW* >ZN29_ASS <SEQ ID 111> MNKIYRIIWNSALNAWVVVSELTRNHTKRASATVETAVLATLLFATVQANATDTDEDDEL EPVVRTAPVLSFHSDKEGTGEKEEVGASSNLTVYFDKNRVLKAGTITLKAGDNLKIKQNT NENTNENTNASSFTYSLKKDLTGLINVETEKLSFGANGKKVNIISDTKGLNFAKETAGTN GDPTVHLNGIGSTLTDTLAGSSASHVDAGNQSTHYTRAASIKDVLNAGWNIKGVKTGSTT GQSENVDFVRTYDTVEFLSADTKTTTVNVESKDNGKRTEVKIGAKTSVIKEKDGKLVTGK GKGENGSSTDEGEGLVTAKEVIDAVNKAGWRMKTTTANGQTGQADKFETVTSGTKVTFAS GNGTTATVSKDDQGNITVKYDVNVGDALNVNQLQNSGWNLDSKAVAGSSGKVISGNVSPS KGKMDETVNINAGNNIEITRNGKNIDIATSMTPQFSSVSLGAGADAPTLSVVEAGALNVG SKDANKPVRITNVAPGVKEGDVTNVAQLKGVAQNLNNRIDNVDGNARAGIAQAIATAGLV QAYLPGKSMMAIGGGTYRGEAGYAIGYSSISDGGNWIIKGTASGNSRGHFGASASVGYQW *

FIG. 8 shows the results of aligning the sequences of each of these strains. Dark shading indicates regions of homology, and gray shading indicates the conservation of amino acids with similar characteristics. As is readily discernible, there is significant conservation among the various strains of ORF 40, further confirming its utility as an antigen for both vaccines and diagnostics.

It will be appreciated that the invention has been described by means of example only, and that modifications may be made whilst remaining within the spirit and scope of the invention.

Appendix 1

Scarlato, Continuation of U.S. App. Ser. No. 10/695,499, filed herewith Ruelle, U.S. Pat. No. 6,780,419 18. (New) An isolated polypeptide comprising 1. An isolated polypeptide comprising a a member selected from the group consisting of member selected from the group consisting of (a) the amino acid sequence of SEQ ID (a) the amino acid sequence of SEQ ID NO: 4; and NO: 2; (b) an immunogenic fragment of at (b) an immunogenic fragment of at least least 15 contiguous amino acids of SEQ ID 15 contiguous amino acids of SEQ ID NO: 2; NO: 4, wherein the immunogenic fragment, wherein the immunogenic fragment, when when administered to a subject in a suitable administered to a subject in a suitable composition which can include an adjuvant, or composition which can include an adjuvant, or a suitable carrier coupled to the polypeptide, a suitable carrier coupled to the polypeptide, induces an antibody or T-cell meditated induces an antibody or T-cell meditated immune response that recognizes the isolated immune response that recognizes the isolated polypeptide SEQ ID NO: 4. polypeptide SEQ ID NO: 2. 19. (New) The isolated polypeptide of claim 2. The isolated polypeptide of claim 1, 18, wherein the polypeptide is according to (a). wherein the polypeptide is according to (a). 20. (New) The isolated polypeptide of claim 3. The isolated polypeptide of claim 1, 18, wherein the polypeptide is according to (b). wherein the polypeptide is according to (b). 21. (New) The isolated polypeptide of claim 4. The isolated polypeptide of claim 1, 18, wherein the immunogenic fragment of (b) wherein the immunogenic fragment of (b) comprises at least 20 contiguous amino acids of comprises at least 20 contiguous amino acids of SEQ ID NO: 4; wherein the immunogenic SEQ ID NO: 2; wherein the immunogenic fragment, when administered to a subject in a fragment, when administered to a subject in a suitable composition which can include an suitable composition which can include an adjuvant, or a suitable carrier coupled to the adjuvant, or a suitable carrier coupled to the polypeptide, induces an antibody or T-cell polypeptide, induces an antibody or T-cell meditated immune response that recognizes the meditated immune response that recognizes the isolated polypeptide SEQ ID NO: 4. isolated polypeptide SEQ ID NO: 2. 22. (New) The isolated polypeptide of claim 5. The isolated polypeptide of claim 1, 18, wherein the isolated polypeptide consists of wherein the isolated polypeptide consists of SEQ ID NO: 4. SEQ ID NO: 2. 23. (New) A fusion protein comprising the 6. A fusion protein comprising the isolated isolated polypeptide of claim 18. polypeptide of claim 1. 24. (New) An immunogenic composition 7. An immunogenic composition comprising comprising the polypeptide of claim 18, and a the polypeptide of claim 1, and a pharmaceutically acceptable carrier. pharmaceutically acceptable carrier. 25. (New) The isolated polypeptide of claim 9. The isolated polypeptide of claim 1, 18, wherein the isolated polypeptide is a wherein the isolated polypeptide is a recombinant polypeptide. recombinant polypeptide. 26. (New) The isolated polypeptide of claim 10. The isolated polypeptide of claim 2, 19, wherein the isolated polypeptide is a wherein the isolated polypeptide is a recombinant polypeptide. recombinant polypeptide. 27. (New) The isolated polypeptide of claim 11. The isolated polypeptide of claim 3, 20, wherein the isolated polypeptide is a wherein the isolated polypeptide is a recombinant polypeptide. recombinant polypeptide. 28. (New) An immunogenic composition 12. An immunogenic composition comprising comprising the isolated polypeptide of claim 19. the isolated polypeptide of claim 2. 29. (New) An immunogenic composition 13. An immunogenic composition comprising comprising the isolated polypeptide of claim 20. the isolated polypeptide of claim 3. 30. (New) A fusion protein comprising the 14. A fusion protein comprising the isolated isolated polypeptide of claim 19. polypeptide of claim 2. 31. (New) A fusion protein comprising the 15. A fusion protein comprising the isolated isolated polypeptide of claim 20. polypeptide of claim 3.

Appendix 2

Written Description Support in the Current Application Written Description (Continuation of Application Support in Application Added Claim # No. 10/695,499) No. PCT/IB99/00103 Claims 18-31 Throughout the Throughout the application and at least at application and at least at the following citations: the following citations: Page 3, lines 2-24; Page 2, line 29 to page 3, Page 31, line 7 to page 34, line 20; line 17; Page 30, line 6 to page Page 52, lines 10-18; 33, line 11; Page 65, line 3 to page 70, Page 50, lines 12-20; line 3. Page 61, line 11 to page 66, line 6. Claims 23, 30, Throughout the Throughout the and 31 application and at least at application and at least at the following citations: the following citations: Page 3, lines 24-27; Page 3, lines 21-24; Page 9, line 26 to page 10, Page 9, lines 11-18; line 4; Page 20, line 6 to page Page 21, lines 1-22. 21, line 4. Claims 25-27 Throughout the Throughout the application and at least at application and at least at the following citations: the following citations: Page 3, lines 24-27; Page 3, lines 17-20; Page 8, line 15 to page 28, Page 8, line 1 to page 27, line 23. line 25.

Appendix 3 Disclosure of Constructive Reductions to Practice within the Scope of the Interfering Subject Matter in Application No. GB 9800760.2, filed Jan. 14, 1998

Number of Amino Acids Location in ORF40 of Location in SEQ 2 in Fragment Application No. GB 9800760.2 of ‘419 Patent 25 Residues 85-109 Residues 127-151 16 Residues 111-126 Residues 153-168 98 Residues 131-228 Residues 173-270 16 Residues 230-245 Residues 272-287 

1-17. (canceled)
 18. An isolated polypeptide comprising a member selected from the group consisting of (a) the amino acid sequence of SEQ ID NO: 4; and (b) an immunogenic fragment of at least 15 contiguous amino acids of SEQ ID NO: 4, wherein the immunogenic fragment, when administered to a subject in a suitable composition which can include an adjuvant, or a suitable carrier coupled to the polypeptide, induces an antibody or T-cell meditated immune response that recognizes the isolated polypeptide SEQ ID NO:
 4. 19. The isolated polypeptide of claim 18, wherein the polypeptide is according to (a).
 20. The isolated polypeptide of claim 18, wherein the polypeptide is according to (b).
 21. The isolated polypeptide of claim 18, wherein the immunogenic fragment of (b) comprises at least 20 contiguous amino acids of SEQ ID NO:4; wherein the immunogenic fragment, when administered to a subject in a suitable composition which can include an adjuvant, or a suitable carrier coupled to the polypeptide, induces an antibody or T-cell meditated immune response that recognizes the polypeptide SEQ ID NO:
 4. 22. The isolated polypeptide of claim 18, wherein the isolated polypeptide consists of SEQ ID NO:
 4. 23. A fusion protein comprising the isolated polypeptide of claim
 18. 24. An immunogenic composition comprising the polypeptide of claim 18, and a pharmaceutically acceptable carrier.
 25. The isolated polypeptide of claim 18, wherein the isolated polypeptide is a recombinant polypeptide.
 26. The isolated polypeptide of claim 19, wherein the isolated polypeptide is a recombinant polypeptide.
 27. The isolated polypeptide of claim 20, wherein the isolated polypeptide is a recombinant polypeptide.
 28. An immunogenic composition comprising the isolated polypeptide of claim
 19. 29. An immunogenic composition comprising the isolated polypeptide of claim
 20. 30. A fusion protein comprising the isolated polypeptide of claim
 19. 31. A fusion protein comprising the isolated polypeptide of claim
 20. 